WO2023198200A1 - Conjugué et composition ainsi que leur procédé de préparation et leur utilisation - Google Patents

Conjugué et composition ainsi que leur procédé de préparation et leur utilisation Download PDF

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WO2023198200A1
WO2023198200A1 PCT/CN2023/088459 CN2023088459W WO2023198200A1 WO 2023198200 A1 WO2023198200 A1 WO 2023198200A1 CN 2023088459 W CN2023088459 W CN 2023088459W WO 2023198200 A1 WO2023198200 A1 WO 2023198200A1
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group
alkyl
conjugate
nucleotide sequence
nucleotides
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WO2023198200A9 (fr
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梁子才
张鸿雁
高山
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苏州瑞博生物技术股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B8/00—Diagnosis using ultrasonic, sonic or infrasonic waves
    • A61B8/08—Detecting organic movements or changes, e.g. tumours, cysts, swellings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/24—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having more than one carboxyl group bound to the carbon skeleton, e.g. aspartic acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C237/06—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atoms of the carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the present disclosure relates to conjugates and pharmaceutical compositions comprising aptamer-based delivery groups and functional groups.
  • the present disclosure also relates to methods of preparation and uses of these conjugates and pharmaceutical compositions.
  • Tumors refer to new organisms formed by the proliferation of local tissue cells in the body under the action of various tumorigenic factors. Among them, the situation in which tumor cells metastasize and invade surrounding tissues is called malignant tumor. According to the classification of tissue cells from which tumors originate, they are generally divided into malignant tumors (cancer) produced by epithelial cells, malignant tumors produced by mesenchymal cells (sarcoma), malignant tumors produced by blood stem cells (leukemia, etc.), and glial cells. Malignant tumors (glioma) produced, etc. Among them, glioma is the most common primary intracranial malignant tumor, accounting for approximately 40%-50% of brain tumors. The global annual incidence rate is 3-8 cases per 100,000 people.
  • gliomas are neuroepithelial tumors, including a variety of pathological types, including but not limited to pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, glioblastoma tumors, oligodendroglioma, anaplastic oligodendroglioma, etc.
  • Aptamers, or nucleic acid aptamers are oligonucleotide molecules that can bind to a variety of target molecules, such as small molecule compounds, proteins, nucleic acids, and even cells, tissues, and organs. Aptamers can provide the important property of "recognition of specific molecules" and are therefore often used in biotechnology and therapy, similar to antibodies. Aptamers can be designed in test tubes and can be quickly synthesized using chemical methods. They also have the excellent properties of being easy to preserve and having low or no immunogenicity. Therefore, they have gradually attracted the attention of researchers in this field in recent years. However, aptamers suitable for tumor-targeted delivery still require further development and application in this field.
  • the inventors of the present disclosure unexpectedly discovered a conjugate that can specifically target tumor cells, especially glioma cells, and the conjugate shows a specific effect on tumor cells, especially glioma cells. Highly specific, it can be effectively enriched in tumor cells, especially glioma cells, and can effectively target tumors. Therefore, the inventor made the following invention:
  • the present disclosure provides a conjugate comprising one or more delivery groups and one or more functional groups; the delivery group consists of an aptamer removing one or more hydrogen atoms Or one or more functional groups are formed, the aptamer includes a continuous nucleotide sequence, and the group connecting two adjacent nucleotides is independently a phosphate group or a phosphate group with a modified group, Each nucleotide is selected from one of modified or unmodified A, U, C or G, and the continuous nucleotide sequence has a sequence represented by formula (1):
  • T 1 is a motif composed of 1-3 nucleotides
  • T 2 is a motif composed of 0-15 nucleotides
  • T 2 does not contain a completely reverse complementary motif to T 1 ;
  • S 1 and S 4 are each a motif composed of 3-7 nucleotides. S 1 and S 4 are the same length and completely reverse complementary;
  • Na and Nc are each a motif consisting of 1-4 nucleotides, each nucleotide in Na is not complementary to every nucleotide in Nc , and in Na and Nc The total number of U accounts for more than 50% of the total number of nucleotides in N a and N c ;
  • S 2 and S 3 are each a motif composed of 1-4 nucleotides. S 2 and S 3 are the same length and completely reverse complementary;
  • N b is a motif composed of 3-6 nucleotides, and the nucleotides at both ends of N b do not form AU or GC complementarity;
  • Each of the delivery groups is independently connected to the functional group via a covalent bond or through a linking group; each of the functional groups is selected from the group consisting of small molecule therapeutic agents that have a therapeutic effect on tumors. group.
  • the present disclosure also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the conjugate of the present disclosure, and a pharmaceutically acceptable carrier.
  • the present disclosure also provides use of the conjugates and/or pharmaceutical compositions of the present disclosure in the preparation of medicaments for the treatment of tumors and tumor-related diseases or symptoms.
  • the present disclosure also provides a method for treating tumors and tumor-related diseases or symptoms, the method comprising administering to a subject in need an effective amount of the conjugate of the present disclosure and/or or pharmaceutical compositions.
  • the present disclosure also provides a kit comprising the conjugates and/or pharmaceutical compositions of the present disclosure.
  • the conjugates and pharmaceutical compositions provided by the present disclosure have excellent ability to target tumors, especially glioma tissues and cells, and can significantly treat or alleviate tumors and tumor-related diseases and/or symptoms.
  • the delivery group in the conjugate provided by the present disclosure can specifically deliver various small molecule drug groups, such as small molecule toxin groups, to tumor tissues and show excellent tumor inhibitory effects.
  • the conjugate of the present disclosure can effectively deliver MMAE to different tumor tissues. While showing tumor targeting ability, it also reduces the risk of toxicity caused by the distribution of MMAE molecules in other tissues, and various administration All methods can effectively inhibit the increase rate of tumor volume and tumor weight, indicating that the conjugate of the present disclosure can effectively inhibit tumor proliferation.
  • further increasing the dosage of the conjugate resulted in almost no increase in tumor volume during the test period, showing a more excellent anti-tumor effect.
  • the inventors of the present disclosure unexpectedly discovered that the conjugates and/or pharmaceutical compositions of the present disclosure can efficiently pass through the blood-brain barrier and can be targeted to gliomas in the brain under systemic administration. , and significantly inhibited the increase in tumor volume and even reduced it to less than 1/10 of the initial volume, and even reduced it to less than 1/100 compared to the control group, indicating that the conjugate of the present disclosure can effectively penetrate the blood-brain barrier and efficiently It targets brain glioma and has a good effect on inhibiting tumor growth, showing good treatment compliance and high drug-making ability to effectively inhibit tumors. .
  • Figures 1A-1C are graphs respectively showing the fluorescence imaging results in mice at 1 hour, 24 hours and 48 hours after administration of different conjugates.
  • Figure 1D shows the results of each group after the mice were sacrificed on D5. Image of fluorescence signals in mouse tumor tissue and kidney.
  • Figures 2A-2C are graphs respectively showing the fluorescence imaging results in mice at 1 hour, 24 hours and 48 hours after administration of different conjugates.
  • Figure 2D is a diagram showing fluorescence signal imaging of tumor tissues and kidneys of mice in each group after the mice were sacrificed on D6.
  • Figure 3 is a line graph showing changes in tumor volume over time in each group of mice after administration of conjugates or control compounds provided by the present disclosure.
  • Figure 4 is a fluorescence imaging diagram showing the brain tissue of mice establishing U118MG orthotopic tumor model after administration of different conjugates at 24h and 48h after administration.
  • Figure 5 is a line graph showing the changes in tumor light intensity values in U118MG orthotopic tumor model mice over time after administration of the conjugates or control compounds provided by the present disclosure.
  • Figure 6 is a line graph showing the changes in tumor volume over time in U118MG subcutaneous tumor model mice after administration of the conjugates or control compounds provided by the present disclosure.
  • Figure 7 is a line graph showing the changes in tumor volume over time in U118MG subcutaneous tumor model mice after administration of different concentrations of conjugates or control compounds provided by the present disclosure.
  • Figure 8 is a line graph showing the changes in tumor volume over time in U118MG subcutaneous tumor model mice after administration of different concentrations of conjugates or control compounds provided by the present disclosure.
  • Figure 9 is a line graph showing the changes in tumor volume over time in A549 subcutaneous tumor model mice after administration of different concentrations of conjugates or control compounds provided by the present disclosure.
  • A, U, C, G and T refer to adenine nucleotides, uracil nucleotides, cytosine nucleotides, guanine nucleotides and thymine nucleotides, respectively.
  • 2-Methylcytosine nucleotide refers to a nucleotide in which the hydrogen at the 2' position of the cytosine base in the cytosine nucleotide is replaced by a methyl group. The structure of these nucleotides is well known to those skilled in the art.
  • nucleic acid motif refers to a fragment of a nucleic acid sequence in an oligonucleotide, consisting of 1 or more nucleotides.
  • a motif is a fragment of a nucleic acid sequence that has a biological function.
  • alkyl refers to straight and branched chains having the specified number of carbon atoms.
  • Chain saturated hydrocarbon radical the number is usually 1 to 20 carbon atoms, such as 1 to 10 carbon atoms, such as 1 to 8 or 1 to 6 carbon atoms.
  • C 1 -C 6 alkyl refers to straight and branched chain alkyl groups containing 1 to 6 carbon atoms.
  • alkyl residue having a particular number of carbons it is intended to encompass all branched and straight chain forms having that number of carbons; thus, for example, "butyl” is meant to include n-butyl, sec-butyl , isobutyl and tert-butyl; “propyl” includes n-propyl and isopropyl.
  • Alkylene is a subset of alkyl and refers to the same residue as alkyl but with two points of attachment.
  • alkenyl refers to an unsaturated branched or linear alkyl group having one or more carbon-carbon double bonds formed by adjacent atoms from the parent alkyl group. Obtained by removing a molecule of hydrogen from a carbon atom. The group can be in the cis or trans configuration of the double bond.
  • alkenyl groups include, but are not limited to: vinyl; propenyl, such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl base), prop-2-en-2-yl; butenyl, such as but-1-en-1-yl, but-1-en-2-yl, 2-methylprop-1-en-1-yl base, but-2-en-1-yl, but-2-en-2-yl, but-1,3-dien-1-yl, but-1,3-dien-2-yl, etc.
  • alkenyl groups have 2 to 20 carbon atoms, and in other embodiments 2 to 10, 2 to 8, or 2 to 6 carbon atoms.
  • Alkenylene is a subset of alkenyl and refers to the same residue as alkenyl but with two points of attachment.
  • alkynyl refers to an unsaturated branched or linear alkyl group having one or more carbon-carbon triple bonds formed by adjacent atoms from the parent alkyl group. Obtained by removing two molecules of hydrogen from a carbon atom.
  • Typical alkynyl groups include, but are not limited to: ethynyl; propynyl, such as prop-1-yn-1-yl, prop-2-yn-1-yl; butynyl, such as but-1-yn- 1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.
  • alkynyl groups have 2 to 20 carbon atoms, while in other embodiments, 2 to 10, 2 to 8, or 2 to 6 carbon atoms.
  • Alkynylene is a subset of alkynyl and refers to the same residue as alkynyl but with two points of attachment.
  • heterocyclyl refers to a stable 3- to 18-membered non-aromatic cyclic group containing 2-12 carbon atoms and 1-6 heteroatoms selected from nitrogen , oxygen and sulfur. Unless otherwise stated in the specification, heterocyclyl is a monocyclic, bicyclic, tricyclic or tetracyclic ring system and may include fused or bridged ring systems. The heteroatoms in the heterocyclyl group may be oxidized heteroatoms. One or more nitrogen atoms, if present, may be quaternized nitrogen atoms. Heterocyclyl groups are partially or fully saturated. A heterocyclyl group can be attached to the rest of the molecule through any ring atom.
  • heterocyclyl groups include, but are not limited to: dioxanyl, thienyl[1,3]disulfonyl (thienyl[1,3]dithianyl), decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindole Dolkyl, 2-oxapiperazinyl, 2-oxapiperidinyl, 2-oxapyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidinonyl, pyrrolidinyl , pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thio
  • aryl refers to a group derived from an aromatic monocyclic or polycyclic hydrocarbon ring system by removal of a hydrogen atom from a ring carbon atom.
  • the aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and 6 to 18 carbon atoms of carbon, wherein one or more rings in the ring system are completely unsaturated, i.e., contain according to Hückel's theory Cyclic, delocalized (4n+2) ⁇ -electron system.
  • Aryl groups include, but are not limited to, phenyl, fluorenyl, and naphthyl groups.
  • Arylene is a subset of aryl and refers to the same residue as aryl but with two points of attachment.
  • Heteroaryl refers to a group derived from a 3- to 18-membered aromatic ring free radical, containing 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur.
  • a heteroaryl group may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, wherein one or more rings in the ring system are fully unsaturated, i.e., contain cyclic ionization according to Hückel's theory. Domain (4n+2) ⁇ -electron system.
  • Heteroaryl groups include fused or bridged ring systems. The heteroatoms in the heteroaryl group may be oxidized heteroatoms.
  • heteroaryl group is attached to the rest of the molecule through any ring atom.
  • heteroaryl groups include, but are not limited to: azepantrienyl, acridinyl, benzimidazolyl, benzindolyl, 1,3-benzobisoxazolyl, benzofuranyl, benzene Oxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, benzo[ b][1,4]oxazinyl (benzo[b][1,4]oxazinyl), 1,4-benzodioxanyl (1,4-benzodioxanyl), benzonaphthofuranyl, benzo Oxazolyl, benzodioxolyl (benzodioxolyl), benzodioxin
  • the present disclosure provides a conjugate comprising one or more delivery groups and one or more functional groups; the delivery group is formed by the aforementioned aptamer removing one or more hydrogen atoms or One or more functional groups are formed; each of the delivery groups is independently connected to the functional group via a covalent bond, or connected through a linking group; each of the functional groups is selected from the group that has the ability to treat tumors The acting small molecule therapeutic agent group.
  • the conjugates of the present disclosure are capable of delivering functional groups to tumors by linking them via covalent bonds or linking groups to form conjugates.
  • the delivery group is formed by removing one or more hydrogen atoms or one or more functional groups from an aptamer
  • the aptamer includes a continuous nucleotide sequence, connecting The groups connecting two adjacent nucleotides are independently phosphate groups or phosphate groups with modified groups, and each nucleotide is selected from one of modified or unmodified A, U, C or G , the continuous nucleotide sequence has the sequence represented by formula (1):
  • T 1 is a motif consisting of 1-3 nucleotides. The inventors found that the presence of T 1 allows the conjugates provided by the present disclosure to display efficient tumor targeting effects.
  • T 1 consists of 2 nucleotides, in which case the conjugate provided by the present disclosure has more excellent tumor targeting ability.
  • T1 consists of 2 nucleotides and contains at least one C.
  • T 1 in the 5'-3' direction, T 1 is CU, UC, or AC.
  • T 2 is a motif consisting of 0-15 nucleotides. The inventors found that T with these nucleotide numbers and various nucleotide sequences did not significantly affect the tumor targeting ability of the conjugates provided by the present disclosure.
  • T2 consists of 0-10 nucleotides. In some embodiments, according to the 5'-3' direction, T2 consists of 1-9 nucleotides starting from U. In this case, the aptamer may have better stability.
  • T 2 does not contain a motif that is completely reverse complementary to T 1 .
  • reverse complementarity refers to the formation of hydrogen bonds between two nucleotide sequences or motifs according to the rules of nucleic acid base pairing, and a nucleotide sequence or motif according to the 5' Each nucleotide in the -3' direction can form base pairing with each nucleotide in the 3'-5' direction of the nucleotide sequence or motif at the other end.
  • "reverse complement” includes one or more of AU, GC, and UG complement.
  • S 1 and S 4 are each a motif composed of 3-7 nucleotides, and S 1 and S 4 are the same length and completely reverse complementary. Aptamers with the above S 1 and S 4 motifs have better stability and can target tumor tissues and cells for a longer period of time.
  • S 1 and S 4 each consist of 3-5 nucleotides and are the same length.
  • GC complementation accounts for more than 40% of the total complementation amount. At this time, the conjugate provided by the present disclosure has further better stability and tumor targeting. ability.
  • S 1 is GCU and S 4 is AGC, or S 1 is GAGU and S 4 is GCUC, or S 1 is GGAGU and S 4 is GCUCU, or S 1 is UAUGG and S 4 is CCAUG.
  • Na and Nc are each a motif consisting of 1-4 nucleotides, and each nucleotide in Na is related to Each nucleotide in N c is not complementary, and the total number of Us in Na and N c accounts for more than 50% of the total number of all nucleotides in Na and N c .
  • Aptamers with the above Na and Nc motifs show excellent tumor tissue targeting ability.
  • the sum of the numbers of nucleotides in Na and Nc is an integer from 2 to 4.
  • the sum of the number of nucleotides in Na and Nc is 3 or 4, and the sum of the number of Us in Na and Nc is 2 or 3.
  • Na and/or Nc are U, UU, UC, or CU in the 5'-3' direction.
  • S 2 and S 3 are each a motif consisting of 1-4 nucleotides. S 2 and S 3 are the same length and are completely reverse complementary. By including S2 and S3 motifs, the conjugates provided by the present disclosure show good stability and excellent tumor targeting ability. In some embodiments, S2 and S3 each consist of 2-3 nucleotides and are the same length. In some embodiments, the reverse complement formed by S 2 and S 3 includes at least one GC complement, in which case the reverse complement has better stability. In some embodiments, in the 5'-3' direction, S2 is CA and S3 is UG, or S2 is AC and S3 is GU, or S2 is GCC and S3 is GGU.
  • N b is a motif consisting of 3-6 nucleotides, and the nucleotides at both ends of N b are not AU or GC complementary. Without being limited by theory, an aptamer with the above N b motif can maintain a specific configuration in space, thereby enabling the conjugate provided by the present disclosure to stably and efficiently target tumor tissues and cells.
  • Nb consists of 4-5 nucleotides.
  • N b in the 5'-3' direction, N b is GACG, GACGU, GACCG, UACU, GUUG, or GAUCU.
  • the inventors of the present disclosure unexpectedly discovered that the delivery group formed by the aptamer having the sequence represented by the above formula (1) can effectively target tumors, especially glioma tissue, thereby allowing the conjugation provided by the present disclosure
  • the drug can specifically enter tumor cells, thereby delivering therapeutic groups more effectively at the cellular level.
  • the length of the continuous nucleotide sequence is 18-50 nucleotides, or 20-40 nucleotides, or 21-36 nucleosides. acid, or 24-32 nucleotides.
  • the delivery group formed by aptamers with these continuous nucleotide lengths and the conjugates provided by the present disclosure including the delivery group can be targeted to tumors more easily, and have advantages in terms of synthesis cost and targeting effect. Good balance.
  • the present disclosure provides conjugates in which the contiguous nucleotide sequence has There is a sequence represented by the following SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3:
  • T 1 is CU
  • S 1 is GCU
  • Na is U
  • S 2 is CA
  • N b GACG
  • S 3 is UG
  • N c is UU
  • S 4 is AGC
  • T 2 is UU;
  • T 1 is CU
  • S 1 is GAGU
  • Na is U
  • S 2 is CA
  • N b is GACG
  • S 3 is UG
  • N c is UU
  • S 4 is GCUC
  • T 2 is U;
  • T 1 is CU
  • S 1 is GAGU
  • Na is U
  • S 2 is CA
  • N b is GACG
  • S 3 is UG
  • N c is UU
  • S 4 is GCUC
  • T 2 is U.
  • the continuous nucleotide sequence has the nucleotide sequence shown in SEQ ID NO: 4:
  • N 1 , N 2 and N 3 are each independently one of A, U, C and G, and N 4 is U, C or G or a motif composed of two of U, C or G; N 5 is U, CU or UU; N 6 is CU, UC or AC; N 7 is U, UU or UUN 8 , and N 8 is a motif composed of 1-15 nucleotides.
  • T 1 is the motif represented by N 6
  • S 1 is the motif represented by GGAGU
  • Na is U
  • S 2 is CA
  • Nb is N 1
  • the motif N 1 N 2 N 3 N 4 composed of N 2 , N 3 and N 4
  • S 3 is UG
  • N c is the motif represented by N 5
  • S 4 is GCUC and the first nucleoside in N 7 Motif composed of acid
  • T 2 is the motif composed of the remaining nucleotides in N 7 .
  • the aptamer containing the nucleotide sequence shown in SEQ ID NO: 4 above can more effectively target tumors, especially gliomas, and be enriched in tumor tissues.
  • the motif N 1 N 2 N 3 N 4 composed of N 1 , N 2 , N 3 and N 4 is one of GACG, GACGU, GACCG, UACU, GUUG or GAUCU, including these motifs
  • the aptamer has higher tumor-specific targeting effect.
  • N 5 is U or UU.
  • the conjugates provided by the present disclosure all have excellent targeting effects on tumors.
  • the aptamer has the nucleotide sequence shown in any one of SEQ ID NOs: 5-11:
  • conjugates provided by the present disclosure having the above nucleotide sequence show a high targeting effect on tumors.
  • motif N8 consists of 1-15 nucleotides. In some embodiments, N8 consists of 1-8 nucleotides.
  • the presence of the motif N 8 makes the conjugates provided by the present disclosure more stable to exonucleases in the body, thereby enabling the tumor-targeting effect to be exerted for a longer period of time in the body.
  • N8 can increase or maintain the tumor targeting effect of the conjugates provided by the present disclosure.
  • the motif N 8 consists of 8 nucleotides.
  • the nucleotide sequence of motif N 8 is CCGAUCUC in the 5'-3' direction.
  • the contiguous nucleotide sequence has the nucleotide sequence shown in any one of SEQ ID NOs: 12-14:
  • the terminal groups at the ribose 3' end of the acid are independently hydroxyl or phosphate groups, and the selection of these terminal groups does not alter the targeting ability of the conjugates provided by the present disclosure.
  • the terminal groups at the 5' end of the ribose sugar of the 5' terminal nucleotide and the 3' end of the ribose sugar of the 3' terminal nucleotide are both hydroxyl groups.
  • each nucleotide may be a modified or unmodified nucleotide.
  • modification of nucleotides may alter the stability and/or tumor targeting capabilities of the conjugates provided by the present disclosure.
  • at least one nucleotide in the conjugates provided by this disclosure is a modified nucleotide.
  • at least one of the groups connecting two adjacent nucleotides in the conjugates provided by the present disclosure has a phosphate group of a modifying group.
  • each of the modified nucleotides is independently a 2′-halogen modified nucleotide, a 2′-alkoxy modified nucleotide, 2 '-alkyl modified nucleotide, 2'-substituted alkyl modified nucleotide, 2'-amino modified nucleotide, 2'-substituted amino modified nucleotide, 2'- One of deoxynucleotides, nucleotides with modified bases, and nucleotide analogs.
  • fluoro-modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribosyl group of the nucleotide is substituted with fluorine and has a structure represented by the following formula (7).
  • Non-fluorinated modified nucleotides refers to nucleotides or nucleotide analogs in which the hydroxyl group at the 2' position of the ribosyl group of the nucleotide is replaced by a non-fluorinated group.
  • each non-fluorinated modified nucleotide is independently selected from nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2' position of the ribosyl group of the nucleotide with a non-fluorinated group. A sort of.
  • Nucleotides formed by replacing the hydroxyl group at the 2' position of the ribosyl group with a non-fluorine group are well known to those skilled in the art. These nucleotides can be selected from 2'-alkoxy modified nucleotides, 2'- Alkyl-modified nucleotides, 2′-substituted alkyl-modified nucleotides, 2′-amino-modified nucleotides, 2′-substituted amino-modified nucleotides, 2′-deoxynucleoside One of the glycosides.
  • the 2'-alkoxy modified nucleotide is a methoxy-modified nucleotide (2'-OMe), as shown in formula (8).
  • the 2'-amino modified nucleotide (2'- NH2 ) is represented by formula (9).
  • 2'-deoxynucleotide (DNA) As shown in equation (10):
  • base modifications include, but are not limited to, adding one or more methyl groups to a base.
  • thymine (T) is considered a type of base-modified uracil (U).
  • 2-methylcytosine is considered a type of base-modified cytosine (C).
  • Nucleotide analogues are those that can replace nucleotides in nucleic acids, but are structurally different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides or thymine deoxyribonucleotides Ribonucleotide group.
  • nucleotide analogs may be isonucleotides, bridged nucleic acid (BNA), or acyclic nucleotides.
  • BNA refers to constrained or inaccessible nucleotides.
  • BNA may contain a five-membered ring, a six-membered ring, or a seven-membered ring bridged structure with a "fixed"C3'-endoglycocondensation. The bridge is typically incorporated into the 2',4'-position of the ribose sugar to provide a 2',4'-BNA nucleotide.
  • BNA can be LNA, ENA, cET BNA, etc., wherein LNA is represented by formula (12), ENA is represented by formula (13), and cET BNA is represented by formula (14):
  • Acyclic nucleotides are a type of nucleotide formed by opening the sugar ring of the nucleotide.
  • the acyclic nucleotide can be unlocked nucleic acid (UNA), glycerol nucleic acid (GNA) or peptide nucleic acid (PNA), wherein UNA is represented by formula (15) and GNA is represented by formula (16) :
  • R is selected from H, OH or alkoxy (O-alkyl).
  • Peptide nucleic acids are a class of nucleotide analogs formed by replacing the glycoside-phosphate backbone with a polypeptide backbone.
  • the peptide nucleic acid may be, for example, a nucleotide analog formed by substituting a 2- aminoethylglycine linkage for a glycoside-phosphate unit.
  • Isonucleotides are compounds formed by changing the positions of the bases on the ribose ring in nucleotides.
  • the isonucleotide can be a compound formed by moving the base from the 1′-position of the ribose ring to the 2′-position or the 3′-position, as shown in formula (17) or (18).
  • Base represents a nucleic acid base, such as A, U, G, C or T; R is selected from H, OH, F or a non-fluorine group as described above.
  • the nucleotide analog is selected from one of isonucleotides, LNA, ENA, cET, UNA, and GNA.
  • each non-fluoro-modified nucleotide is a methoxy-modified nucleotide, above and below, the methoxy-modified nucleotide refers to the 2' of the ribosyl group -Nucleotides formed by replacing the hydroxyl group with a methoxy group.
  • each cytosine nucleotide in the contiguous nucleotide sequence in the conjugates provided by the present disclosure is a fluoro-modified cytosine nucleotide, and/or the Each uracil nucleotide in the contiguous nucleotide sequence is a fluorinated modified uracil nucleotide.
  • each nucleotide in the contiguous sequence of nucleotides in the conjugates provided by the present disclosure is a 2'-methoxy modified nucleotide.
  • the present disclosure provides conjugates in which one or more uracil nucleotides have modified bases.
  • a thymine base (T) is considered a uracil base (U) with a methyl modification.
  • the group connecting two adjacent nucleotides can be a phosphate group or a modified phosphate group.
  • the modification of the phosphate group is, for example, replacing at least one non-bridging oxygen atom in the phosphate group with a sulfur atom to form a phosphorothioate group or a phosphorodithioate group.
  • at least one group connecting two adjacent nucleotides in the conjugates provided by the present disclosure is a phosphorothioate group.
  • at least one of the three groups connecting two adjacent nucleotides between the first four nucleotides at the 5' end of the continuous nucleotide sequence is a phosphorothioate Ester group.
  • At least 2 of the 3 groups connecting two adjacent nucleotides between the first four nucleotides at the 5' end of the continuous nucleotide sequence are phosphorothioates. Ester group. In some embodiments, at least one of the groups connecting two adjacent nucleotides between the first four nucleotides at the 3' end of the continuous nucleotide sequence is a phosphorothioate group. In some embodiments, at least 2 of the 3 groups connecting two adjacent nucleotides between the first four nucleotides at the 3' end of the continuous nucleotide sequence are phosphorothioates. Ester group. In some embodiments, each group connecting two adjacent nucleotides in the contiguous nucleotide sequence is a phosphorothioate group.
  • the conjugate provided by the present disclosure with the above modifications is not only low in cost, but also makes it difficult for ribonucleases in the body to cleave the linking group, thereby increasing the stability of the conjugate provided by the present disclosure and making it more resistant. Nuclease hydrolysis performance.
  • the conjugate provided by the present disclosure including the above-mentioned modified delivery group has higher activity of targeting tumor tissue and/or cells.
  • the contiguous nucleotide sequence has a nucleotide sequence set forth in one of SEQ ID NOs: 15-39:
  • the capital letters C, G, U, and A represent the base composition of nucleotides;
  • the small letter m means that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide;
  • the small letter f means The adjacent nucleotide to the left of the letter f is a fluorinated modified nucleotide;
  • the lowercase letter s indicates that the two nucleotides to the left and right of the letter s are connected by a phosphorothioate group.
  • the present disclosure provides a conjugate having a structure represented by formula (101):
  • each R AP group is independently a group having a structure shown in formula (2):
  • each AP group is the same or different and independently represents one of the delivery groups; each A 0 group is the same or different and independently represents one of the functional groups; R j , each R k Or each R i is the same or different, each independently represents a covalent bond or a connecting group, and both R i and R k are different at the same time as a covalent bond; m 0 is an integer from 1 to 6; n 0 is 1- An integer of 6, each n 1 independently represents an integer of 0-4; Indicates the site at which a group is covalently attached.
  • m 0 is an integer of 1-6, that is, the conjugate represented by formula (101) contains 1-6 functional groups A 0 .
  • m 0 is an integer of 1-4, that is, the conjugate represented by formula (101) contains 1-4 functional groups A 0 .
  • m 0 is 1, that is, the conjugate represented by formula (101) contains 1 functional group A 0 .
  • n 0 is an integer from 1 to 6, that is, the conjugate represented by formula (101) contains 1 to 6 R AP groups. From the perspective of delivery efficiency and cost, in some embodiments, n 0 is an integer of 1-3, that is, the conjugate represented by formula (101) contains 1-3 R AP groups. In some embodiments, n 0 is 1, that is, the conjugate represented by formula (101) contains 1 R AP group.
  • each n 1 independently represents an integer from 0 to 4, and both Ri and Rk are not covalently bonded at the same time, thus each R AP group contains 1 to 5 delivery groups. AP. In some embodiments, each n 1 independently represents an integer from 0 to 1, such that each R AP group contains 1 to 2 delivery groups AP. In some embodiments, n 0 is 1 and n 1 is 0. In this case, the conjugate represented by formula (101) contains 1 delivery group AP.
  • the function of R k and R i is to covalently connect the delivery group AP to the R j group and to the functional group A 0 via the R j group. Therefore, any R k or R i that can achieve the above connection without negatively affecting the effects of the delivery group AP and the functional group A 0 can be used in the present invention.
  • each n 1 is 0, and each R i is independently a covalent bond, or one of the following linking groups
  • One or more connection combinations C 1 -C 20 alkylene, phosphate bond, phosphorothioate bond, amide bond, ester bond, ether bond, thioether bond, disulfide bond, 1, 2, 3- Triazole subunit, polyethylene glycol subunit, pyrrolidine subunit, 2-oxopyrrolidine subunit, phenylene, cyclohexylene, 2-succinimide subunit, 2-thiobutanediyl Imide subunit, amino acid subunit, nucleotide subunit.
  • the linking group Rj includes linking groups known to those skilled in the art to be useful in antibody drug conjugates.
  • the linking group R j may be cleavable or non-cleavable.
  • linking group Rj may be cleavable.
  • "cleavable" means that upon targeting of the conjugates of the present disclosure to a tumor, the linker group Rj undergoes covalent cleavage in the intratumoral environment and/or within the tumor cells, releasing the individual therapeutic The agent group produces the therapeutic effect.
  • the linker R j comprises an activating enzyme linker, a sulfatase-cleavable linker, a galactose-cleavable linker, a lysosomal protease-sensitive linker, a peptidyl linker group, a glucuronide linking group, an acid-sensitive cleavable linking group, or a glutathione-sensitive disulfide linking group.
  • linking group R j comprises a peptidyl linking group.
  • the peptidyl linking group is selected from the group consisting of valine-citrulline dipeptide linker (Val-Cit), alanine-alanine dipeptide linker (Ala-Ala), valine - One or more of the alanine dipeptide linker (Val-Ala) and the glycine-glycine-phenylalanine-glycine tetrapeptide linker (Gly-Gly-Phc-Gly).
  • linking group R j is selected from N-succinimide 4-(2-dithiopyridine)butyrate (SPDB), N-succinimide-4-(2-thiopyridine) Subunit) valerate (SPP), (S)-2-((S)-2-amino-3-methylbutanamide)-5-ureidopentanoic acid (Val-Cit-PAB-OH), N -Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) or 2-(phosphate-(CH 2 ) 6 -S-)- One of the maleimidocaproyl-valine-citrulline-p-aminobenzyl subunits.
  • SPDB N-succinimide 4-(2-dithiopyridine)butyrate
  • SPP N-succinimide-4-(2-thiopyridine) Subunit) valerate
  • SPP S)-2-((S)-2-amino
  • the linking group R j includes the linking groups listed in McKertish CM, Kayser V. Advances and Limitations of Antibody Drug Conjugates for Cancer. Biomedicines. 2021 Jul 23; 9(8): 872. The entirety of this document is incorporated by reference. The content is incorporated into this article in its entirety.
  • the linking group R j includes a valine-citrulline dipeptide linker (Val-Cit), a polyethylene glycol subunit, an iminohexyl subunit, an N-succinimidyl group, One or more of the GAU trinucleotide linking groups.
  • each R i is independently a covalent bond, a disulfide bond, a dodecylene group, a valine-citrulline dipeptide linker (Val-Cit), a polyethylene glycol subunit , iminohexyl subunit, N-succinimidyl group or GAU trinucleotide subunit or a connection combination of two.
  • the function of the R j group is to connect the R AP group with the functional group A 0 , thereby specifically delivering the functional group A 0 to the tumor through the tumor targeting effect of the delivery group AP in the R AP group.
  • Tumor tissue and/or cells Tumor tissue and/or cells. Therefore, any R j group that can achieve the above connection without affecting the tumor targeting effect of the delivery group AP and the effect of the functional group A 0 can achieve the purpose of the present invention and solve the technology to be solved by the present invention. question.
  • the conjugate represented by formula (1) after the conjugate represented by formula (1) reaches the tumor tissue and/or enters the tumor cell, the R j is cleaved, releasing the pharmaceutically active molecule corresponding to the separate functional group A 0 .
  • the R j does not undergo cleavage in vivo, and the presence of the R j group and the R AP group in the conjugate will not affect the therapeutic effect of the functional group A 0 .
  • R j is a covalent bond
  • m 0 is 1.
  • the conjugate represented by formula (101) contains 1 functional group A 0 and 1 R AP group, each R AP groups are directly linked to the functional group A 0 .
  • each R AP group is linked to the same atom of functional group A0 .
  • each R AP group is attached to a different atom of functional group A0 .
  • R j is a linking group comprising a backbone moiety, a side chain moiety , and a conjugation linkage.
  • each side chain moiety is independently a covalent bond, or a linear alkylene group of 1 to 70 carbon atoms in length, or one or more of the linear alkylene groups
  • each conjugation linkage is independently a covalent bond or a combination of one or more of the following linkage structures: C 1 -C 10 linear alkylene, phosphate ester bond, phosphorothioate Ester bond, amide bond, ester bond, ether bond, disulfide bond, 1,2,3-triazole subunit, polyethylene glycol subunit, pyrrolidine subunit, 2-oxopyrrolidine subunit, phenylene base, cyclohexylene group, 2-succinimide subunit, 2-thiosuccinimide subunit, amino acid subunit, nucleotide subunit.
  • each of the conjugated linking parts in the linking group R j is respectively connected to the main chain part and one of the functional groups A 0 ; the number of side chain parts is n 0 , Each side chain moiety is respectively linked to the backbone moiety and one of the R AP groups.
  • each functional group A 0 and R AP group is each independently linked to the linking group R j .
  • all side chain moieties are connected to the same atom in the backbone moiety; alternatively, each side chain moiety is connected to a different atom in the backbone moiety.
  • m 0 is 1, and the linking group R j includes a structure shown in formula (301):
  • k is an integer from 1 to 3; LC is the main chain part, L A is the side chain part, LB is the conjugation connection part, Indicates the site at which a group is covalently attached.
  • the length of LC is 5-30 atoms, wherein the length of LC refers to the longest atomic chain in LC formed from the atoms directly connected to LA to the atoms directly connected to LB The number of atoms in the chain. To simplify the structure, in some embodiments, the length of LC is 8-25 atoms.
  • the conjugated linker LB is a connection combination of one or more of the following 1-5 linkages: phosphate bond, phosphorothioate bond, amide bond, ester bond, ether bond, and disulfide bond.
  • k is an integer of 1-3;
  • L C contains any one of the groups represented by formula (L1)-(L3), through the group represented by formula (L1)-(L3) The ether bond in the group is connected to the L A part:
  • L C contains a group represented by formula (L1), and the O atom in group (L1) is directly connected to L A.
  • L B is a phosphate bond or disulfide bond
  • Each L A is a covalent bond, or each L A is selected from the group consisting of groups (L4)-(L23) and their connecting combinations:
  • each j1 is an integer from 1 to 10;
  • Each R' is C 1 -C 10 alkyl
  • Each Ra is a hydrogen atom, a C 1 -C 10 alkyl group, or selected from the group consisting of groups (L24) - (L37):
  • each LA is a connected combination of at least 2 of the groups (L4)-(L9), (L13), (L14), (L18). In some embodiments, each LA is a connected combination of at least 2 of the groups (L4), (L5), (L7), (L9), (L13), (L14), (L18).
  • L A has an amide bond-containing structure as shown in Formula (302)
  • L B has an N-acylpyrrolidine-containing structure as shown in Formula (303), containing a carbonyl group and an oxygen atom
  • L C is a linking group based on hydroxymethylaminomethane, dihydroxymethylaminomethane or trimethylolaminomethane:
  • n 302 , q 302 and p 302 are each independently an integer from 2 to 6.
  • n 302 , q 302 and p 302 are each independently an integer from 2 or 3;
  • n 303 is an integer from 4 to 16.
  • Select the location, n 303 is an integer from 8 to 12, Indicates the site at which a group is covalently attached.
  • each of the side chain moieties LA is associated with one R AP group via It is connected by a phosphate bond, an ether bond or an ester bond, and is connected by the oxygen atom of the hydroxyl group in the main chain part LC forming an ether bond with the main chain part LC ;
  • the conjugated connection part LB is connected by the formula
  • the carbonyl group in (303) is connected to the nitrogen atom of the amino group in the main chain part L C to form an amide bond, and the oxygen atom in formula (303) forms a phosphate bond or ether bond with the functional group A 0 bond or ester bond.
  • the backbone moiety LC is a linking group based on hydroxymethylaminomethane, dihydroxymethylaminomethane or trimethylolaminomethane via the oxygen atom of the hydroxyl group.
  • Each of the side chain moieties L A is connected by an ether bond and is connected to the conjugate linker L B by an amide bond via the nitrogen atom of the amino group.
  • 1-3 side chains in the linking group Rj are connected to the carbon atoms of the same aminomethyl group and are connected to the R AP group containing the delivery group through the conjugated linker LB.
  • the conjugate has the structure shown in formula (305):
  • the linking group R j comprises a structure shown in formula (306):
  • n 306 is an integer from 0 to 3
  • each p 306 is independently an integer from 1 to 6,
  • the linkage combination formed by all pyrrolidin subunits and any possible phosphodiester groups constitutes the backbone part, consisting of the carbonyl group attached to the nitrogen atom of the pyrrolidin subunit and the one marked by *
  • a chain of atoms between the oxygen atoms shown constitutes each side chain moiety, and the side chain part is connected to the R AP group by an ether bond formed by the oxygen atom marked by *;
  • at least one of the oxygen atoms marked by # To conjugate the linking part and form an ether bond, ester bond or phosphate bond with the functional group A 0 , and the remaining oxygen atoms marked by # are connected to hydrogen atoms to form hydroxyl groups, or with C 1 -C 3 alkyl groups The connection forms a C 1 -C 3 alkoxy group.
  • the present disclosure provides conjugates having a structure shown in formula (307a), (307b) or (307c):
  • the present disclosure provides conjugates having a structure shown in formula (308):
  • n 308 can be an integer from 1 to 10; in some embodiments, n 308 can be an integer from 2 to 6, taking into account various aspects such as ease of synthesis, structure/process cost, and tumor cell specificity. In some embodiments, n 308 is 3 or 4.
  • Each R 3 is independently a functional group A 0 , or a R AP group including a delivery group AP;. In some embodiments, at least one R3 is the functional group A0 , and at least one R3 is RAP . In some embodiments, one R 3 is a functional group A 0 and the remaining R 3 are R AP groups.
  • each m 308 when each m 308 is independently selected from an integer of 2-10, it is considered possible to make the spatial positions between the multiple delivery groups AP in the conjugate more suitable for contact with the surface of tumor cells. The corresponding receptors interact.
  • each m 308 is independently an integer of 2-5, In some embodiments, each m 308 is equal.
  • each R 308 is independently selected from H, C 1 -C 10 alkyl, C 1 -C 10 haloalkyl, and C 1 -C 10 alkoxy, formula (308) does not change The properties of the conjugates indicated are suitable for achieving the purposes of this disclosure.
  • each R 308 is independently selected from H, methyl, or ethyl.
  • each R 308 is H.
  • Each L 1 connected to the functional group A 0 represents the conjugation linkage, and each L 1 connected to the R AP represents the side chain moiety.
  • one R 3 is the functional group A 0 and the remaining R 3 are the R AP group.
  • one or more L 1 serves as the side chain moiety connecting the R AP group to an N atom on the nitrogen-containing backbone; and an additional one or more L 1 serves as the conjugation linker , connect the functional group A 0 to the N atom on the nitrogen-containing skeleton.
  • the nitrogen-containing skeleton together constitutes the main chain part of the connecting group Rj .
  • nitrogen-containing backbone refers to the chain structure in the structure represented by formula (308), in which the carbon atom to which R 308 is attached and the N atom are interconnected.
  • each L 1 is independently 3-25 atoms.
  • each L1 is independently 4-15 atoms in length.
  • L1 is defined as a linear alkylene group for convenience, it may not be a linear group or have a different name, such as an amine or alkenyl group resulting from the above substitutions and/or substitutions.
  • the length of L is the number of atoms in the chain connecting two junction points.
  • the ring obtained by replacing the carbon atoms of the linear alkylene group (such as heterocyclylene or heteroarylene) is calculated according to the minimum number of atoms between the connection points on the ring corresponding to the ring in the chain. The length of the section.
  • L 1 is selected from the group consisting of the groups represented by the above formulas L4-L23 and any connected combinations thereof. In some embodiments, each L 1 is independently selected from the group consisting of linked combinations of at least 2 of groups L4-L9, L13, L14, L18. In some embodiments, each L 1 is independently a connected combination of at least 2 of the groups L4, L5, L7, L9, L13, L14, and L18.
  • each L 1 simultaneously contains a connection site connected to an N atom on the nitrogen-containing skeleton and a functional group A 0 or the R
  • the connection site where the AP group is connected to the N atom on the nitrogen-containing skeleton forms an amide bond with the N atom.
  • one or more L1 is selected from B5, B6, B5' or B6':
  • q 2 is an integer from 1 to 10. In some embodiments, q2 is an integer from 1 to 5.
  • each R AP group contains multiple functional groups.
  • each functional group in the compound of formula (308) is the same functional group.
  • each functional group in the compound of formula (308) is a functional group serving the same purpose and function.
  • the compound of formula (308) contains different types of functional groups for different purposes and functions.
  • the compound represented by formula (308) has formula (403), (404), (405), (406), (407), (408), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418), (419), (420), (421), (422), (423 ), (424), (425), (426) or (427):
  • linking group R j comprises nucleotide sequence I and nucleotide sequence II, each of said nucleotide sequence I and said nucleotide sequence II comprising 5-25 modified or unmodified Nucleotides, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary, the delivery group is connected to the nucleotide sequence I, and the functional group is connected to the Nucleotide sequence II, and said nucleotide sequence I and said nucleotide sequence II do not trigger an immune response or toxic reaction in a subject.
  • the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary or completely reverse complementary; or, the nucleotide sequence I and the nucleotide sequence II are The lengths are equal and both are 10-20 modified or unmodified nucleotides; alternatively, the nucleotide sequence I and the nucleotide sequence II are both composed of 17 nucleotides and are completely reverse complementary.
  • the 3' end of the delivery group is connected to the ribose 5' position of the 5' terminal nucleotide of the nucleotide sequence I via a phosphate bond
  • the functional group is connected to the The ribose 5' position of the 5' terminal nucleotide of the nucleotide sequence II;
  • the functional group includes a nucleotide sequence, the 3' end of the nucleotide sequence is connected to the The ribose sugar 5' position of the 5' terminal nucleotide of the nucleotide sequence I.
  • the nucleotide sequence I and the nucleotide sequence II have the sequences shown in SEQ ID NO: 40 and SEQ ID NO: 41 respectively:
  • nucleotide sequence I and the nucleotide sequence II respectively have Sequences shown in SEQ ID NO:42 and SEQ ID NO:43:
  • the functional group is a small molecule therapeutic agent group that has a therapeutic effect on tumors, and each small molecule therapeutic agent group is independently selected from a cytotoxic group, an antibiotic group, or an angiogenesis inhibitory group. agent.
  • the conjugates of the present disclosure can specifically deliver the small molecule therapeutic agent group to the tumor, thereby affecting the disease process or symptoms of the tumor through the action of the small molecule therapeutic agent group.
  • cytotoxic groups for example, by specifically delivering cytotoxic groups to tumors through the conjugates of the present disclosure, causing specific death of cancer cells in the tumors, thereby reducing the low-targeting band of the cytotoxin itself. While causing side effects, it can significantly reduce the number of cancer cells in the tumor, thereby treating the tumor.
  • the small molecule therapeutic agent group is formed by removing one or more hydrogen atoms or one or more functional groups from the following small molecule therapeutic agent: the small molecule therapeutic agent is selected from the group consisting of methotrexate, Mycins, vinca alkaloids, auristatin (including MMAE and MMAF), calicheamicin, maytansine, camptothecin, and calicheamicin.
  • the small molecule therapeutic agent group is a group formed by removing one or more hydrogen atoms or one or more functional groups from monomethyl auristatin E (MMAE).
  • MMAE monomethyl auristatin E
  • the functional groups may be included in the conjugates of the present disclosure by any suitable means.
  • the functional group A 0 can be connected to the main chain moiety through the aforementioned conjugation linker.
  • the conjugates provided by the present disclosure further comprise one or more delivery aid groups selected from the group consisting of C 10 -C 30 hydrocarbon groups, cholesterol groups, and phospholipid groups. one or more.
  • the conjugates provided by the present disclosure are better compatible with the in vivo environment in the central nervous system, may have better bioavailability, and/or make the conjugates provided by the present disclosure more compatible with the in vivo environment in the central nervous system. Deliver drugs to tumors more efficiently.
  • the delivery aid group is linked to the delivery group or the linking group via a covalent bond or linking group. In some embodiments, the delivery aid group is linked to the functional group.
  • the synthesis method of the conjugate provided by the present disclosure includes contacting the protected conjugate with a deprotection reagent in a solvent under deprotection reaction conditions, and isolating to obtain the conjugate provided by the present disclosure.
  • a protected conjugate is a compound in which any active functional group in the conjugate provided by the present disclosure is protected by a protecting group.
  • the reactive functional groups include, but are not limited to, hydroxyl, amino and/or phosphate groups, and the protecting groups are, respectively, hydroxyl protecting groups, amino protecting groups and/or phosphate hydroxyl protecting groups (e.g., cyanoethyl base protecting group).
  • the solvent, deprotection reaction conditions and deprotection reagents used are selected and determined.
  • the deprotection reaction conditions, solvents and deprotection reagents are those used in solid phase synthesis of nucleic acids.
  • the method includes adding the protected conjugate into a mixed solution of methylamine aqueous solution and ammonia water, and the deprotection reaction conditions include reaction at normal temperature and pressure for 1-5 h.
  • the methylamine aqueous solution and the saturated concentrated ammonia solution are mixed in equal volumes to obtain the mixed solution, and the dosage of the solution relative to the protected conjugate is 0.1-10 ml/ ⁇ mol.
  • the separation includes purification by column chromatography, collecting the product eluate and removing the solvent.
  • the purification conditions may be, for example, using a preparative ion chromatography purification column and eluting with a gradient eluent of sodium chloride aqueous solution and sodium phosphate aqueous solution.
  • the conjugate provided by the present disclosure has a structure represented by formula (101), and the synthesis method of the protected conjugate includes in an organic solvent, under coupling reaction conditions, will contain active
  • the compound of the group R One or more hydrogen atoms or one or more functional groups are formed, and each of the functional groups is independently a small molecule therapeutic agent group that has a therapeutic effect on tumors, wherein the delivery group and the functional group Any reactive group of is protected by a protecting group, and the reactive group R x1 and the reactive group R x2 are groups capable of forming a covalent bond or connecting group R j through reaction.
  • the delivery group to which the reactive group R x1 is attached and the delivery group to which The molar ratio of the functional groups of the active group R x2 is m 0 :n 0 .
  • the active groups in the delivery group and functional group include, but are not limited to, one or more of hydroxyl, amino, and phosphate groups, and the protective group is correspondingly a hydroxyl protective group. , one or more of amino protecting group, phosphate hydroxyl protecting group (for example, cyanoethyl protecting group).
  • the compound containing the reactive group R x1 and the delivery group can obtain compounds containing the reactive group R x1 and the delivery group through various methods.
  • the compound containing the reactive group R Obtained through synthesis.
  • the compound containing the reactive group R The sequence of nucleotides in the chain is used to connect the nucleoside monomers in sequence, where at least one nucleoside monomer is a nucleoside monomer with an active group R
  • the phosphoramide solid-phase synthesis method connects a phosphoramidite monomer or a protected phosphoramidite monomer with an active group R x1 , and then removes the protecting group to form an active group R x1 .
  • the coupling reaction conditions are condensation reaction conditions or thiol-disulfide exchange reaction conditions.
  • the coupling reaction conditions are condensation reaction conditions, and the condensation reaction conditions are acylation condensation reaction conditions, dehydration condensation reaction conditions or click chemical reaction conditions, and the reactive group R x1 and the reactive group R x2 is a group capable of undergoing the aforementioned condensation reaction.
  • the condensation reaction conditions are conditions for an acylation condensation reaction, and the active groups R x1 and R x2 are groups capable of undergoing an acylation condensation reaction to form R I .
  • the condensation reaction conditions are conditions for dehydration condensation reaction, one of the active groups R x1 and R x2 is a group containing an acid halide group or a carboxyl group, and the other is a group containing an amino group or a hydroxyl group. group.
  • the condensation reaction conditions are click chemistry conditions, one of the reactive groups R x1 and R x2 is a group containing an alkynyl group, and the other is a group containing an azide group.
  • the condensation reaction conditions are Michael addition reaction conditions, one of the active groups R x1 and R x2 is a group containing a thiol group, and the other is a group containing a thiol group.
  • the condensation reaction conditions are N-hydroxysuccinimide-carbodiimide (NHS-EDC) coupling reaction conditions, and one of the active groups R x1 and R x2 One is a group containing N-hydroxysuccinimide (NHS), and the other is a group containing a carbodiimide group (EDC).
  • NHS-EDC N-hydroxysuccinimide-carbodiimide
  • EDC carbodiimide group
  • the compound containing the reactive group R x1 and the delivery group is prepared by contacting an aptamer with the reactive group R x0 and a cross-linking agent under the conditions of a coupling reaction,
  • the cross-linking agent contains click chemical active groups and acylation groups.
  • the reactive group R is an active ester group, for example, it can be one of an NHS ester group, an imide ester group, and a pentafluorophenyl ester group.
  • the cross-linking agent can be done as follows: Michael E., et al.
  • the reactive group RxO is amino.
  • the coupling conditions are basic conditions.
  • the alkaline condition is a condition in which a weak alkali aqueous solution is present, such as a sodium bicarbonate aqueous solution.
  • the aptamer with the active group R x0 is synthesized by synthesizing the aptamer. It is prepared by using phosphoramidite monomers containing active groups at corresponding positions in the process. Those skilled in the art can obtain phosphoramidite monomers containing reactive groups in various ways.
  • the reactive group R The monomer may be 6-(trifluoroacetylamino)-hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite monomer which is easily commercially available, wherein the reactive group R x0 is an amino group, and the active group R x0 can be connected to the phosphoramidite monomer through a phosphoramidite solid phase synthesis method.
  • the oligonucleotide is single-stranded, it is obtained by removing the trifluoroacetyl protecting group through a deprotection reaction (such as ammonolysis of concentrated ammonia) that is easily implemented by those skilled in the art.
  • the coupling reaction condition is one of sulfhydryl-disulfide bond exchange reactions, one of the reactive groups R x1 and R Sulfur bonded leaving group.
  • R x1 in the above-mentioned phosphoramidite monomer containing reactive group R x1 exists in the form of protected R x1 ', and the preparation method also includes deprotection reaction conditions Next, the step of isolating the compound containing the active group R x1 and the delivery group by contacting the prepared compound containing the protected active group R x1 ' and the delivery group with a deprotecting reagent.
  • the disulfide bond activator is a disulfide pyridine.
  • phosphorus acid amide monomer containing the reactive group R Amide monomers are commercially available, for example, phosphoramidite monomers represented by formula (105) can be obtained commercially.
  • n 105 and m 105 are each independently an integer from 1 to 10.
  • the coupling reaction conditions are thiol- disulfide bond exchange reaction conditions, the reactive group R
  • the skilled person can obtain it in various known ways, for example, by using a phosphoramidite monomer containing a thiol group, prepared by a phosphoramidite solid phase synthesis method, or obtained commercially.
  • the coupling reaction conditions are phosphoramidite solid phase synthesis reaction conditions
  • the reactive group R x2 is a phosphoramidite group
  • the reactive group R x2 and a functional group include
  • the compound may be, for example, a readily commercially available compound containing a phosphoramidite group and a small molecule therapeutic agent group.
  • the coupling reaction conditions are Michael addition reaction conditions
  • the reactive group R x2 is an N-succinimide group
  • the compound containing the reactive group R x2 and a functional group can be Examples include readily commercially available compounds containing N-succinimide groups and small molecule therapeutic agent groups.
  • the reactive group R x1 and the reactive group R x2 are nucleotide sequence I and nucleotide sequence II respectively, and the nucleotide sequence I and the nucleotide sequence II each comprise 5-25 modified or unmodified nucleotides, said nucleotide sequence I and said nucleotide sequence II being at least partially reverse complementary, said delivery group being linked to said nucleotide sequence I,
  • the functional group is connected to the nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II do not trigger an immune response or toxic reaction in the subject, and the coupling reaction conditions It is the reaction condition for annealing to form nucleic acid double strands.
  • nucleotide sequence I and the nucleotide sequence II each consist of 17 nucleotides and are completely reverse complementary. In some embodiments, the nucleotide sequence I and the nucleotide sequence II have the sequences shown in SEQ ID NO: 40 and SEQ ID NO: 41, respectively. In some embodiments, the nucleotide sequence I and the nucleotide sequence II have the sequences shown in SEQ ID NO: 42 and SEQ ID NO: 43, respectively.
  • the delivery group is formed by the removal of one or more hydrogen atoms or functional groups by the aptamer.
  • the ribose 5′ group of the 5′ terminal nucleotide and the ribose 3′ group of the 3′ terminal nucleotide of the continuous nucleotide sequence are both hydroxyl groups, and the delivery group is formed by an aptamer at The delivery group is formed by removing a hydrogen atom from the 5' hydroxyl group of the 5' terminal nucleotide; in some embodiments, the delivery group is formed by the aptamer removing a hydrogen atom from the 3' hydroxyl group of the 3' terminal nucleotide; in some In embodiments, the delivery group is formed by an aptamer removing the 5' hydroxyl group from the 5' terminal nucleotide; in some embodiments, the delivery group is formed by an aptamer removing the 3' hydroxyl group from the 3' terminal nucleotide.
  • the delivery group is formed by the aptamer removing the ribose 2'-hydroxyl group from the nucleotide it contains.
  • the aptamer can be obtained by conventional aptamer preparation methods in the art (such as nucleic acid solid-phase synthesis and liquid-phase synthesis methods). Among them, nucleic acid solid-phase synthesis already has commercial customization services.
  • Modified nucleotide groups can be introduced into the conjugates provided by the present disclosure by using nucleoside monomers with corresponding modifications, methods of preparing nucleoside monomers with corresponding modifications, and modifying nucleotide groups. Methods for introducing aptamers are also well known to those skilled in the art. All modified nucleoside monomers are commercially available or prepared using known methods.
  • the conjugates may also be used in the present disclosure in the form of pharmaceutically acceptable salts or precursor compounds thereof.
  • pharmaceutically acceptable salt refers to the formation of a drug into a corresponding salt that does not produce additional pharmaceutical side effects on the human body in order to increase the stability, solubility and/or bioavailability of the drug, such as potassium salt, sodium salt, carboxylate, etc.
  • Previous “Body compound” refers to a compound that, although structurally and functionally identical to the conjugate itself, is capable of reacting to form the conjugate provided by the present disclosure after entering the body or in a body fluid environment, thereby Compounds that exert effects and achieve the purposes provided by this disclosure.
  • these precursor compounds can increase the stability of the drug, extend the sustained release time, increase bioavailability, etc.
  • the precursor compounds The compound includes a precursor group that can react in the human body to form all functional groups A 0 in the conjugate.
  • the precursor compound includes all active hydroxyl groups in the conjugate substituted with acetoxy groups to form
  • the precursor compound includes a prodrug group formed from a precursor compound of the therapeutic agent corresponding to the functional group in the conjugate. residue.
  • the prodrug group may be, for example, a group formed by replacing the active hydrogen in the hydroxyl or amino functional group in the functional group with an acyl group, an alkyl group, or a phosphoryl group.
  • the present disclosure also provides a pharmaceutical composition comprising the conjugate provided by the present disclosure and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier can be a carrier commonly used in the art, such as but not limited to water, physiological saline, magnetic nanoparticles (such as nanoparticles based on Fe 3 O 4 or Fe 2 O 3 ), carbon Nanotubes (carbon nanotubes), mesoporous silicon (mesoporous silicon), calcium phosphate nanoparticles (calcium phosphate nanoparticles), polyethylenimine (PEI), polyamide dendrimer (polyamidoamine (PAMAM) dendrimer), Poly(L-lysine), PLL, chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) , poly(D&L-lactic/glycolic acid)copolymer (PLGA), poly(2-aminoethyl ethylene phosphate) (poly(2-aminoethyl ethylene phosphate), PPEEA) and one or more of poly(2-dimethylaminoethy
  • the pharmaceutically acceptable carrier contains a physiologically acceptable compound that acts, for example, to stabilize the pharmaceutical composition or to increase or decrease the absorption of the conjugate and/or the pharmaceutical composition.
  • the physiologically acceptable compound is selected from one or more of the following compounds: carbohydrates, such as glucose, sucrose and/or dextran; antioxidants, such as ascorbic acid and/or glutathione; chelating agents; Molecular weight proteins; compositions that reduce clearance or hydrolysis of any co-administered substances; excipients; stabilizers and buffers. Detergents may also be used to stabilize the composition or to increase or decrease absorption of the pharmaceutical composition.
  • the physiologically acceptable compound may also include one or more wetting agents, emulsifiers, dispersants or preservatives, especially to prevent the growth or action of microorganisms.
  • the physiologically acceptable compounds are known to those skilled in the art and will not be described in detail in this disclosure. As will be readily understood by those skilled in the art, the selection of pharmaceutically acceptable carriers and physiologically acceptable compounds depends, for example, on the route of administration and the specific physiochemical properties of any co-administered substances.
  • pharmaceutically acceptable carriers are sterile and generally free of undesirable materials.
  • the pharmaceutical composition provided by the present disclosure may further contain pharmaceutically acceptable auxiliary substances as needed to approximate physiological conditions, such as pH adjusters and buffers, toxicity adjusters, etc., such as sodium acetate, sodium chloride, potassium chloride, chloride Calcium, sodium lactate, etc., the concentration of the conjugate provided by the present disclosure in the pharmaceutical composition can vary within a wide range, and is mainly selected according to a specific administration method based on fluid volume, viscosity, body weight, etc.
  • the pharmaceutical composition has no special requirements on the content of the conjugate and the pharmaceutically acceptable carrier.
  • the weight ratio of the conjugate to the pharmaceutically acceptable carrier can be It is 1: (1-500). In some embodiments, the above weight ratio is 1: (1-50).
  • the pharmaceutical composition may also contain other pharmaceutically acceptable auxiliary materials, which may be one or more of various preparations or compounds commonly used in the art.
  • the other pharmaceutically acceptable excipients may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator.
  • the pH buffer can be a trishydroxymethylaminomethane hydrochloride buffer with a pH of 7.5-8.5 and/or a phosphate buffer with a pH of 5.5-8.5, for example, it can be a phosphate with a pH of 5.5-8.5. Buffer.
  • the protective agent may be at least one of myo-inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose and glucose. Based on the total weight of the pharmaceutical composition, the content of the protective agent may be 0.01-30% by weight.
  • the osmotic pressure regulator may be sodium chloride and/or potassium chloride.
  • the content of the osmotic pressure regulator is such that the osmotic pressure of the pharmaceutical composition is 200-700 milliosmole/kg (mOsm/kg).
  • the content of the osmotic pressure regulator can be easily determined by those skilled in the art based on the desired osmotic pressure.
  • the dosage of the preparation made from the pharmaceutical composition during administration will be adjusted due to different administration methods.
  • the pharmaceutical composition can be a liquid preparation, such as an injection; it can also be a freeze-dried powder injection, which is mixed with liquid excipients during administration to prepare a liquid preparation.
  • the liquid preparation can be used for, but is not limited to, subcutaneous, intramuscular or intravenous injection, and the pharmaceutical composition can also be delivered by, but not limited to, puncture injection, oropharyngeal inhalation, or nasal administration.
  • the pharmaceutical composition is for subcutaneous, intramuscular, intravenous or intrathecal administration.
  • the pharmaceutical composition may be in the form of a liposome formulation.
  • the pharmaceutically acceptable carrier used in the liposome formulation includes an amine-containing transfection compound (hereinafter also referred to as an organic amine), a helper lipid, and/or a pegylated Lipids.
  • an organic amine, auxiliary lipid and pegylated lipid can be respectively selected from the amine-containing transfection compounds described in Chinese patent application CN103380113A (which is incorporated by reference into this disclosure in its entirety) or One or more of its pharmaceutically acceptable salts or derivatives, auxiliary lipids and pegylated lipids.
  • the organic amine can be a compound represented by formula (201) described in Chinese patent application CN103380113A or a pharmaceutically acceptable salt thereof:
  • X 101 and X 102 are each independently O, S, NA or CA, where A is hydrogen or a C 1 -C 20 hydrocarbon chain;
  • R 101 , R 102 , R 103 , R 104 , R 105 , R 106 and R 107 are each independently hydrogen, cyclic or acyclic, substituted or unsubstituted, branched or linear aliphatic group Group, cyclic or acyclic, substituted or unsubstituted, branched or linear heteroaliphatic group, substituted or unsubstituted, branched or linear acyl group, substituted or unsubstituted Substituted, branched or linear aryl, substituted or unsubstituted, branched or linear heteroaryl;
  • x is an integer from 1 to 10;
  • n is an integer from 1 to 3
  • m is an integer from 0 to 20
  • R 103 and nitrogen in formula (201) form a structure shown in formula (202) or formula (203):
  • g, e and f are each independently an integer from 1 to 6
  • HCC represents a hydrocarbon chain
  • each *N represents a nitrogen atom in formula (201).
  • R 103 is a polyamine. In other embodiments, R 103 is a ketal. In some embodiments, each of R 101 and R 102 in formula (201) is independently any substituted or unsubstituted, branched or linear alkyl or alkenyl group, the alkyl The radical or alkenyl group has 3 to about 20 carbon atoms, such as 8 to about 18 carbon atoms, and 0 to 4 double bonds, such as 0 to 2 double bonds.
  • R 103 may be any of the following formulas (204) to (213):
  • g, e and f are each independently an integer from 1 to 6
  • each "HCC” represents a hydrocarbon chain
  • each * shows that R 103 is the same as in formula (201)
  • Possible attachment points to the nitrogen atoms in where each H at any * position can be replaced to effect attachment to the nitrogen atom in formula (201).
  • the compound represented by formula (201) can be prepared according to the description in Chinese patent application CN103380113A.
  • the organic amine is an organic amine represented by formula (214) and/or an organic amine represented by formula (215):
  • the auxiliary lipid is cholesterol, cholesterol analogs and/or cholesterol derivatives
  • the pegylated lipid is 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine-N-[methoxy (polyethylene glycol)]-2000.
  • the molar ratio between the organic amine, the auxiliary lipid and the PEGylated lipid is (19.7-80): (19.7-80 ):(0.3-50), for example, it can be (50-70):(20-40):(3-20).
  • the pharmaceutical composition particles formed from the conjugates provided by the present disclosure and the above-described amine-containing transfection reagent have an average diameter of about 30 nm to about 200 nm, typically about 40 nm to about 135 nm, more typically, the liposome particles have an average diameter of about 50 nm to about 120 nm, about 50 nm to about 100 nm, about 60 nm to about 90 nm, or about 70 nm to about 90 nm, for example, the liposome particles The average diameter is about 30, 40, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150 or 160 nm.
  • the conjugate in a pharmaceutical composition formed from a conjugate provided by the present disclosure and the above-mentioned amine-containing transfection reagent, is PEGylated with all lipids (e.g., organic amines, auxiliary lipids, and/or
  • the weight ratio (weight/weight ratio) of lipids) is from about 1:1 to about 1:50, from about 1:1 to about 1:30, from about 1:3 to about 1:20, from about 1: 4 to about 1:18, from about 1:5 to about 1:17, from about 1:5 to about 1:15, from about 1:5 to about 1:12, from about 1:6 to about 1:12 Or in the range from about 1:6 to about 1:10, for example, the weight ratio of the conjugate provided by the present disclosure to all lipids is about 1:5, 1:6, 1:7, 1:8, 1 ⁇ 9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17 or 1:18.
  • the weight ratio of the conjugate provided by the present disclosure to all lipids is
  • each component of the pharmaceutical composition may exist independently when sold, and may exist in the form of a liquid preparation when used.
  • the pharmaceutical composition formed by the conjugate provided by the present disclosure and the above-mentioned pharmaceutically acceptable carrier can be prepared according to various known methods, except that the existing adapter is replaced by the conjugate provided by the present disclosure.
  • the body or conjugate is sufficient; in some embodiments, it can be prepared as follows:
  • the organic amine, auxiliary lipid and PEGylated lipid are suspended in alcohol according to the above molar ratio and mixed to obtain a lipid solution; the amount of alcohol is such that the total mass concentration of the obtained lipid solution is 2-25 mg/mL, For example, it can be 8-18 mg/mL.
  • the alcohol is selected from pharmaceutically acceptable alcohols, such as alcohols that are liquid near room temperature, for example, ethanol, propylene glycol, benzyl alcohol, glycerin, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400 One or more, for example, it can be ethanol.
  • the conjugate provided by the present disclosure is dissolved in a buffered salt solution to obtain an aqueous conjugate solution.
  • concentration of the buffer salt solution is 0.05-0.5M, for example, it can be 0.1-0.2M.
  • the pH of the buffer salt solution is adjusted to 4.0-5.5, for example, it can be 5.0-5.2.
  • the amount of the buffer salt solution is such that the concentration of the conjugate does not exceed 0.6mg/mL, for example, it can be 0.2-0.4mg/mL.
  • the buffer salt is selected from one or more of soluble acetate and soluble citrate, for example, it can be sodium acetate and/or potassium acetate.
  • the volume ratio of the lipid solution and the aqueous conjugate solution is 1:(2-5), for example, it can be 1:4.
  • the incubated liposome preparation is concentrated or diluted, impurities are removed, and sterilization is performed to obtain the pharmaceutical composition provided by the present disclosure.
  • Its physical and chemical parameters are a pH value of 6.5-8, an encapsulation rate of not less than 80%, and a particle size of 40-200nm, polydispersity index not higher than 0.30, osmotic pressure 250-400mOsm/kg; for example, the physical and chemical parameters can be pH value 7.2-7.6, encapsulation rate not less than 90%, particle size 60-100nm, and more
  • the dispersion index is not higher than 0.20, and the osmotic pressure is 300-400mOsm/kg.
  • concentration or dilution can be performed before, after or at the same time as impurities are removed.
  • Various existing methods can be used to remove impurities, such as using a tangential flow system, a hollow fiber column, ultrafiltration at 100K Da, and the ultrafiltration exchange solution is phosphate buffer saline (PBS) with pH 7.4.
  • PBS phosphate buffer saline
  • the sterilization method can adopt various existing methods, for example, filtration sterilization can be performed on a 0.22 ⁇ m filter.
  • the present disclosure also provides use of the conjugates and/or pharmaceutical compositions provided by the present disclosure in the preparation of medicaments for the treatment of tumors and tumor-related diseases or symptoms.
  • the present disclosure also provides a method of treating tumors and tumor-related diseases or symptoms, the method comprising administering to a subject in need thereof a conjugate and/or pharmaceutical combination described in the present disclosure. things.
  • the methods of the present disclosure can effectively treat tumors and tumor-related diseases or symptoms; and, under the highly specific targeting effect of the conjugates provided by the present disclosure, Distribution of therapeutic agents to undesirable other body organs/tissues can be reduced, reducing potential side effects.
  • radiotherapy and/or chemotherapy drugs that are commonly used in the field of tumor treatment and are known to have significant side effects, it is of great significance and significant value.
  • administering/administering refers to the administration of a conjugate and/or pharmaceutical composition by a method or pathway that at least partially localizes the conjugate and/or pharmaceutical composition to a desired site to produce the desired effect. or the pharmaceutical composition is placed into a subject.
  • Routes of administration suitable for the methods of the present disclosure include local administration and systemic administration. Generally speaking, local administration results in the delivery of more conjugates and/or pharmaceutical compositions to a specific site compared to the subject's entire body; whereas systemic administration results in the delivery of all conjugates and/or pharmaceutical compositions to the entire body of the subject.
  • the conjugate and/or pharmaceutical composition is delivered to substantially the entire body of the subject.
  • the inventors of the present disclosure unexpectedly discovered that the conjugates and/or pharmaceutical compositions of the present disclosure can efficiently pass through the blood-brain barrier and can be targeted to tumors in the brain when administered systemically. This further improves the delivery efficiency of functional groups, saves costs and reduces undesirable side reactions.
  • Administration to the subject may be by any suitable route known in the art, including, but not limited to, oral or parenteral routes, such as intravenous, intramuscular, subcutaneous, or transdermal. medicine, airway administration (aerosol), pulmonary administration, nasal administration, rectal administration and topical administration (including buccal administration and sublingual administration).
  • the frequency of administration may be once or more daily, weekly, every two weeks, every three weeks, monthly or yearly.
  • the dosage of the conjugates and/or pharmaceutical compositions described in the present disclosure can be conventional dosages in the art, and the dosage can be determined according to various parameters, especially the age, weight and gender of the subject. Toxicity and efficacy can be measured in cell culture or experimental animals by standard pharmaceutical procedures, such as determining the LD50 (the dose that causes 50% of the population to be lethal) and the ED50 (the dose that causes 50% of the maximum response intensity in a quantitative response, and in a qualitative response Medium refers to the dose that causes a positive reaction in 50% of the experimental subjects). Dosage ranges for humans can be derived based on data from cell culture assays and animal studies.
  • the conjugates are administered to The amount of the conjugate in the conjugate and/or pharmaceutical composition: the amount of the conjugate can be 0.001-100 mg/kg body weight, in some embodiments 0.01-50 mg/kg body weight, in further embodiments It is 0.05-20 mg/kg body weight, in a further embodiment it is 0.1-15 mg/kg body weight, and in still further embodiment it is 0.1-10 mg/kg body weight.
  • the above amounts may be preferred when administering the conjugates and/or pharmaceutical compositions of the present disclosure.
  • the present disclosure provides a kit comprising a conjugate and/or pharmaceutical composition provided by the present disclosure.
  • kits described in the present disclosure may provide the conjugate and/or pharmaceutical composition in one container.
  • the kits described in the present disclosure may include a A container providing pharmaceutically acceptable excipients.
  • the kit may also include other ingredients, such as stabilizers or preservatives.
  • a kit of the present disclosure can include at least one additional therapeutic agent in a container other than the container in which the conjugates and/or pharmaceutical compositions of the present disclosure are provided.
  • the kit may contain instructions for mixing the conjugate and/or pharmaceutical composition with pharmaceutically acceptable carriers and/or excipients or other ingredients, if any.
  • the conjugate and pharmaceutically acceptable carrier and/or excipients and the pharmaceutical composition, and/or pharmaceutically acceptable excipients can be provided in any form, such as liquid form, dry form form or lyophilized form.
  • the conjugate and pharmaceutically acceptable carrier and/or excipients and the pharmaceutical composition and optional pharmaceutically acceptable excipients are substantially pure and/or sterile.
  • sterile water can be provided in the kits of the present disclosure.
  • the reagents and culture media used in the following examples are all commercially available products.
  • the nucleic acid electrophoresis, real-time PCR and other operations used are all described in Molecular Cloning (Cold Spring Harbor LBboratory Press (1989)). method to proceed.
  • the conjugates numbered AP1-AP8 and AP18 in Table 1 were synthesized by solid-phase synthesis method, and the nucleotide sequences corresponding to AP1-AP8 and AP18 in Table 1 were followed from the 3′-5′ direction. After connecting all the nucleoside monomers, connect the Cy5 phosphoramidite monomer (purchased from Suzhou Jima Company, batch number CY5P21H1B) according to the solid-phase synthesis method to connect the nucleoside phosphoramidite monomers. Subsequently, the nucleotide sequence was added to a mixed solution of equal volumes of methylamine aqueous solution and ammonia water.
  • the dosage of the solution relative to the oligonucleotide was 0.5 ml/ ⁇ mol.
  • the reaction was carried out at 25°C for 2 hours.
  • the solid was removed by filtration, and the supernatant was vacuumed. Concentrate to dryness.
  • eluent A 20mM sodium phosphate (pH 8.1)
  • eluent B 1.5M sodium chloride, 20mM sodium phosphate (pH 8.1)
  • the specific conditions include using a Sephadex column for desalting.
  • the filler is Sephadex G25 and eluting with deionized water.
  • the obtained eluate was concentrated to remove the solvent and lyophilized to obtain conjugates AP1-AP8 and AP18 in which the 5' position of the ribose sugar of the 5' terminal nucleotide was connected to the fluorescent group Cy5 through a phosphate linking group, respectively.
  • the prepared conjugates are diluted to a concentration of 0.2 using ultrapure water (made by Milli-Q ultrapure water instrument, resistivity 18.2M ⁇ *cm (25)) mg/mL, use liquid chromatography-mass spectrometry (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters Company, model: LCT Premier) for molecular weight detection.
  • ultrapure water made by Milli-Q ultrapure water instrument, resistivity 18.2M ⁇ *cm (25)
  • LC-MS liquid chromatography-mass spectrometry
  • the capital letters C, G, U, and A indicate the base composition of nucleotides; the small letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; the small letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; f means that the nucleotide adjacent to the left side of the letter f is a fluorinated modified nucleotide; CY5 means The connection site of the fluorescent dye group Cy5 (Cyanine 5) group on the aptamer.
  • the capital letters C, G, U, and A represent the base composition of nucleotides; the small letter m represents that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; MMAE represents The connection site of the small molecule drug group MMAE (monomethyl auristatin E) group on the aptamer.
  • conjugate 20 was prepared according to the following steps.
  • the dye group in conjugate AP2 was replaced by the small molecule drug group MMAE, and the linking group was 2-(phosphate Ester-(CH 2 ) 6 -S-)-maleimidocaproyl-valine-citrulline-p-aminobenzyl subunit.
  • the oligonucleotide sequence in conjugate 20 in Table 2 was synthesized by solid-phase synthesis method. The only difference is that the nucleoside monomers are connected in sequence according to the oligonucleotide sequence corresponding to conjugate 20 in Table 2; During the phase synthesis process, after connecting the last nucleoside monomer at the 5' end, follow the method of connecting nucleoside phosphoramidite monomers, and additionally connect HO-(CH 2 ) 6 -SS-(CH 2 ) 6 - group phosphoramidite monomer (purchased from Hongene Biotech Company), cleave the oligonucleotide single chain from the solid support to obtain the oligonucleotide single chain S1 shown in formula (20-a) ( 70.00mg, 6.42 ⁇ mol):
  • TCEP tris (2-chloroethyl) phosphate, 0.37 mmol, purchased from Bi A TCEP aqueous solution obtained by dissolving the medicine (batch number: BD155793) in 10.0 ml of purified water. Mix well and react at room temperature for 2 hours. Dilute the reaction solution with 10 mL of purified water and filter to obtain 28 mL of reaction solution. Transfer the reaction solution to a 3K ultrafiltration tube and centrifuge at 3900 rpm for 30 min. Repeat the steps of ultrafiltration and centrifugation twice more to collect the products in the filter membrane to obtain oligonucleotide S2 (67.0 mg, yield: 95.7%).
  • the obtained crude conjugate 20 was diluted with 0.5 ml of purified water and filtered with a 0.45 ⁇ m membrane.
  • the filtrate was purified using Agilent semi-preparative reversed-phase column chromatography.
  • the product peak eluate was collected, and the solvent was evaporated to remove conjugate 20 (55 mg, yield 56.7%).
  • the molecular weight was detected by LC-MS, the theoretical value: 12092.73, the measured value: 12091.39, the measured value is consistent with the theoretical value, indicating that the conjugate 20 has the structure shown in S3, and the conjugate 20 combines the dye in the conjugate AP2
  • the group is replaced with the small molecule drug group MMAE, and the linking group is 2-(phosphate-(CH 2 ) 6 -S-)-maleimidocaproyl-valine-citrulline-p Aminobenzyl subunit (2-(phosphate-(CH 2 ) 6 -S-)-MC-Val-Cit-PAB).
  • This experimental example examines the targeting properties of the prepared conjugates AP2, AP3-8 and comparison AP9-contrast AP12 in mice.
  • U118MG human colloid was cultured in DMEM complete medium (MACGENE Company, Cat. No. CM15019) supplemented with 10% fetal bovine serum (FBS, RMBIO Company) at 37 in an incubator containing 5% CO 2 /95% air.
  • Tumor cells purchased from Guangzhou Genio Biotechnology Co., Ltd.).
  • mice 24 NOD-SCID mice (purchased from Spefford (Beijing) Biotechnology Co., Ltd.), female, 12 weeks old.
  • the above cell culture medium was inoculated into the subcutaneous position of the right forelimb of NOD-SCID mice.
  • the inoculation volume was 100 ⁇ L per mouse, that is, 1 ⁇ 10 7 cells were inoculated into each mouse. After injection, the mice were continued to be raised for 20 days to obtain mice inoculated with U118MG subcutaneous tumors.
  • the aptamers AP2, AP3-8 and comparison AP9-comparison AP12 prepared above were prepared into 0.3 mg/mL solutions using DMEM medium.
  • Administration began 14 days after U118MG cells were inoculated, and the day of administration was recorded as D1.
  • mice inoculated with U118MG subcutaneous tumors were randomly divided into 12 groups, with 2 mice in each group:
  • mice For 7 groups of mice, AP2, AP3, AP4, AP5, AP6, AP7 or AP8 were administered to each mouse in each group respectively.
  • the volume of single administration was 10 ⁇ L/g mouse body weight. Calculation shows that single administration The dose is 3mg/kg, which are recorded as test groups 1A-1G respectively;
  • each mouse in each group was given contrast AP9, contrast AP10, contrast AP11 or contrast AP12 respectively.
  • the single administration volume was 10 ⁇ L/g mouse body weight.
  • the single administration dose was calculated. 3mg/kg, respectively recorded as control group 1H-1K;
  • each mouse was given DMEM culture medium with a dosage volume of 10 ⁇ L/g mouse body weight, which was recorded as the blank control group 1Y.
  • each mouse was imaged in vivo using the small animal in vivo optical imaging system IVIS Lumina Series III. On D5, mice in each group were sacrificed and tumor tissues and kidneys were harvested for fluorescence imaging.
  • FIGs 1A-1C show, respectively, 1h, 24h and 48h after administration of different conjugates. Picture of the fluorescence imaging results in mice. The leftmost one among the three mice in each small picture is the mouse in the blank control group 1Y. As can be seen from Figure 1A, the blank control group did not show any fluorescence signal; unlike this, 1 hour after administration, the mice in each test group and the control group showed fluorescence signals at the subcutaneous tumors; as can be seen from Figures 1B and 1C, At 24h and 48h after administration, only the mice in the test group 1A-1G showed strong fluorescence signals at the subcutaneous tumors, while the mice in the control group 1H-1K showed almost no fluorescence signals or only very weak ones. Fluorescent signal.
  • Figure 1D is a diagram showing the fluorescence signal imaging of tumor tissues and kidneys of mice in each group after the mice were sacrificed on D5, where Blank represents the blank control group 15Y.
  • Blank represents the blank control group 15Y.
  • the tumor tissues of the mice in the blank control group 1Y and the control group 1H-1K showed almost no fluorescence signal or only a very weak fluorescence signal; in contrast, the test group 1A given the conjugate of the present disclosure
  • the tumor tissues of the -1G mice all showed strong fluorescence signals, while only weak fluorescence signals were shown at the metabolic organ kidney, indicating that compared with the control conjugate, the delivery group in the conjugate provided by the present disclosure is included.
  • the conjugates of the group can target tumor tissues more stably and efficiently.
  • This experimental example examines the targeting properties of the prepared conjugates AP2, AP1 and comparison AP13-contrast AP17 in mice.
  • U118MG human colloid was cultured in DMEM complete medium (MACGENE Company, Cat. No. CM15019) supplemented with 10% fetal bovine serum (FBS, RMBIO Company) at 37 in an incubator containing 5% CO 2 /95% air.
  • Tumor cells purchased from Guangzhou Genio Biotechnology Co., Ltd.).
  • mice 16 NOD-SCID mice (purchased from Spefford (Beijing) Biotechnology Co., Ltd.), male, 12 weeks old.
  • the above cell culture medium was inoculated into the subcutaneous position of the right forelimb of NOD-SCID mice.
  • the inoculation volume was 100 ⁇ L per mouse, that is, 1 ⁇ 10 7 cells were inoculated into each mouse. After injection, the mice were continued to be raised for 20 days to obtain mice inoculated with U118MG subcutaneous tumors.
  • the aptamers AP2, AP1 and comparison AP13-contrast AP17 prepared above were prepared into 0.3 mg/mL solutions using DMEM medium.
  • Administration began 21 days after U118MG cells were inoculated, and the day of administration was recorded as D1.
  • mice inoculated with U118MG subcutaneous tumors were randomly divided into 8 groups, with 2 mice in each group:
  • mice For the two groups of mice, AP2 or AP1 was administered to each mouse in each group, and the single administration volume was 10 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 3 mg/kg, which were recorded in sequence as Test Group 2A-2B;
  • each mouse in each group was given contrast AP13, contrast AP14, contrast AP15, contrast AP16, or contrast AP17.
  • the volume of a single dose was 10 ⁇ L/g mouse body weight. The calculation showed that a single dose The dosage was 3 mg/kg, which were recorded as control group 2C-2G respectively;
  • each mouse was given DMEM culture medium with a dosage volume of 10 ⁇ L/g mouse body weight, which was recorded as the blank control group 2Y.
  • each mouse was imaged in vivo using the small animal in vivo optical imaging system IVIS Lumina Series III. On D6, mice in each group were sacrificed and tumor tissues and kidneys were harvested for fluorescence imaging.
  • FIGs 2A-2C are respectively diagrams showing the fluorescence imaging results in mice at 1h, 24h and 48h after administration of different conjugates, in which the leftmost one of the three mice in each small picture is It is the blank control group of 2Y mice.
  • the blank control group did not show any fluorescence signal; unlike this, 1 hour after administration, the mice in each test group and the control group showed fluorescence signals at the subcutaneous tumors; as can be seen from Figures 2B and 2C, At 24h and 48h after administration, only the mice in test groups 2A and 2B showed strong fluorescence signals at the subcutaneous tumors, while the mice in the blank control group 2Y and the control groups 2C-2G showed no fluorescence signals at all.
  • Figure 2D is a diagram showing the fluorescence signal imaging of tumor tissues and kidneys of mice in each group after the mice were sacrificed on D6, where Blank represents the blank control group 2Y.
  • Blank represents the blank control group 2Y.
  • the tumor tissues of the mice in the blank control group 2Y and the control group 2C-2G showed no fluorescent signal at all; in contrast, the tumor tissues of the mice in the test group 2A or 2B were administered with the conjugate of the present disclosure.
  • aptamers with various sequences shown in formula (1) can stably and efficiently Targeting tumor tissue further demonstrates that the conjugates provided by the present disclosure containing delivery groups formed by these aptamers can effectively reach tumor tissue.
  • This experimental example examined the anti-tumor activity of the prepared conjugate 20 in mice.
  • mice in this experiment were purchased from Spefford Company.
  • the germ line is NOD-SCID, the grade is SPF, both genders are female, and the age is 6-8 weeks.
  • U118MG glioma cells were purchased from Genio.
  • U118MG cells growing in the logarithmic phase were digested and resuspended in DMEM complete medium (MACGENE Company, Cat. No. CM15019) supplemented with 10% fetal bovine serum (FBS, GIBCO Company) and cultured until the cell density was 1 ⁇ 10 8 cells/mL to obtain a culture medium containing U118MG cells.
  • the above culture medium containing U118MG cells was inoculated into the subcutaneous position of the right forelimb of each mouse, with an injection volume of 100 ⁇ L. Thus, each mouse was inoculated with 1 ⁇ 10 7 U118MG glioma cells.
  • the conjugate AP2 prepared above was prepared into a 1.94 mg/mL solution using PBS.
  • the day of cell inoculation was recorded as D1, and the cells were administered once each on D8, D12, D16 and D20.
  • mice 36 mice were randomly divided into the following 6 groups, with 6 mice in each group:
  • PBS was administered by tail vein injection, with a single administration volume of 10 ⁇ L/g;
  • control group 3b the above-mentioned conjugate AP2 solution was administered by tail vein injection, with a single administration volume of 10 ⁇ L/g and a single administration dose of 15.5 mg/kg;
  • MMAE solution was administered by tail vein injection, with a single administration volume of 10 ⁇ L/g and a single administration dose of 0.3 mg/kg;
  • the conjugate 20 solution with the above concentration of 0.625 mg/mL was administered by tail vein injection, with a single administration volume of 10 ⁇ L/g and a single administration dose of 5 mg/kg (based on oligonucleotide mass basis) containing MMAE at a dose equivalent to 0.3 mg/kg;
  • the conjugate 20 solution with the above concentration of 2.06 mg/mL was administered by tail vein injection, with a single administration volume of 10 ⁇ L/g and a single administration dose of 16.5 mg/kg. (based on oligonucleotide mass) containing MMAE at a dose equivalent to 1 mg/kg;
  • the conjugate 20 solution with the above concentration of 1.25 mg/mL was administered by subcutaneous injection, with a single administration volume of 5 ⁇ L/g and a single administration dose of 5 mg/kg (based on the mass of the oligonucleotide). ) containing MMAE at a dose equivalent to 0.3 mg/kg.
  • the long diameter and short diameter of the tumor were determined by in vitro measurement. Tumor volume was calculated according to the formula 1/2 (long diameter ⁇ short diameter 2 ). Before the first administration on D8, the tumor volume of each group was measured and the average tumor volume was recorded. The tumor volume of each group was measured and recorded on D16, twice a week.
  • Figure 3 is a line graph showing changes in tumor volume over time in each group of mice. It can be seen from the results in Figure 3 that in the blank control group 3a and control group 3b that were only given PBS and AP2, the tumor volume increased rapidly; in the control group 3c that was only given MMAE, the tumor volume increase rate was reduced, indicating that MMAE itself has an effect on tumor proliferation. Shows inhibitory effect. Furthermore, test groups 3d and 3f, whose MMAE content was equivalent to that of the control group 3c, had significantly smaller tumor volumes during the test period than the control group 3c, showing more excellent anti-tumor activity than the control group 3c given MMAE alone. This shows that the conjugate provided by the present disclosure can effectively deliver MMAE to tumor tissue.
  • tumor targeting ability While showing tumor targeting ability, it also reduces the risk of toxicity caused by the distribution of MMAE molecules in other tissues, and various administration All methods can effectively inhibit tumor proliferation.
  • the tumor volume of test group 3e which further increased the dosage, hardly increased during the test period, showing a more excellent anti-tumor effect.
  • U118MG human colloid was cultured in DMEM complete medium (MACGENE Company, Cat. No. CM15019) supplemented with 10% fetal bovine serum (FBS, RMBIO Company) at 37 in an incubator containing 5% CO 2 /95% air.
  • Tumor cells purchased from Guangzhou Genio Biotechnology Co., Ltd.).
  • the cells were digested with 0.25wt% trypsin and collected. The supernatant was aspirated and the cells were resuspended in DMEM medium supplemented with 10% FBS to prepare a cell culture medium with a cell density of 4 ⁇ 10 7 cells/mL.
  • NOD-SCID mice purchased from Spefford (Beijing) Biotechnology Co., Ltd.
  • the above cell culture fluid was inoculated into NOD-SCID mice, and the cell culture fluid was injected into the right striatum of the mouse using the lateral cerebral ventricle injection method.
  • the position was AP (anteroposterior anteroposterior/anterior position): 1 mm
  • ML medial lateral
  • DV diorsal ventral
  • injection volume 10 ⁇ L that is, each mouse is inoculated with 4 ⁇ 10 5 cells.
  • the mice were kept for 14 days after injection.
  • AP2 and comparative AP19 were dissolved into conjugate solutions with a concentration of 0.3 mg/mL (based on aptamer) using 1 ⁇ DMEM medium. Take 4 of the aforementioned mice and administer them through tail vein injection. Inject AP2 and comparative AP19 solutions respectively. The dosage of all animals is calculated according to their body weight. The dosage volume is 10 ⁇ L/g. Based on the amount of aptamer, each The animal dosage was 3 mg/kg, and each group was administered to 2 mice, which were recorded as test group 4a and control group 4b respectively.
  • mice in each group were killed and the brain tissue was taken.
  • mice were killed and the brain tissue was taken.
  • the mouse brain tissue was analyzed in IVIS Lumina Series III. Fluorescence imaging. The results are shown in Figure 4.
  • Figure 4 is a fluorescence imaging diagram showing the brain tissue of mice establishing U118MG orthotopic tumor model after administration of blank control group 4Y, test group 4a and control group 4b at 24h and 48h after administration.
  • Blank represents the blank control group
  • Ith represents intrathecal injection
  • iv represents tail vein injection.
  • U118MG human glioma cells expressing the Luciferase (Photinus pyralis) reporter gene were cultured according to the method of Experimental Example 4, hereinafter referred to as U118MG-luc human glioma cells (purchased from Nanjing Jingkebai Biotechnology Co., Ltd.). Take the U118MG-luc human glioma cells growing in the logarithmic phase, digest them with 0.25wt% trypsin and collect the cells. After centrifugation, aspirate the supernatant and resuspend the cells in serum-free DMEM medium to make the cell density: Cell culture medium of 4 ⁇ 10 7 cells/mL.
  • mice 24 Balb/C-nude nude mice (purchased from Spefford (Beijing) Biotechnology Co., Ltd.), male, 12 weeks old.
  • the above cell culture fluid was inoculated into the striatum of Balb/C-nude nude mice, and the mouse striatum injection method was used to inject the cell culture fluid into the right striatum of the mouse at the AP (anteroposterior/posterior/anterior) position. position): 1 mm, ML (medial lateral): 1.5 mm, DV (dorsal ventral): 3.5 mm, injection volume 10 ⁇ L, that is, each mouse is inoculated with 4 ⁇ 10 5 cells. After in situ tumor inoculation, the mice were continued to be fed for 14 days.
  • Conjugate 20 was dissolved into a conjugate solution with a concentration of 1 mg/mL (based on aptamer) using 1 ⁇ DMEM (purchased from Zhongke Maichen (Beijing) Technology Co., Ltd., batch number: K1902200) medium.
  • Conjugate 21 and comparative conjugate 24 were respectively dissolved into conjugate solutions with a concentration of 0.8 mg/mL (based on aptamer).
  • the small animal in vivo optical imaging system IVIS Lumina Series III was used to conduct in vivo imaging of each mouse. According to the brain fluorescence intensity, there were 6 mice in each group. The day of administration was recorded as D1 (i.e. , the first day of the experiment, the following D4, D8, etc. correspond to the fourth day, the eighth day of the experiment, and so on).
  • In vivo imaging method Each mouse was intraperitoneally injected with 10 ⁇ L/g body weight concentration of 15 mg/mL D-luciferin potassium salt working solution (purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.), and in vivo imaging was performed 10 min after injection ( Lumina III small animal in vivo imaging system). After imaging, circle the fluorescence region (ROI) of the mouse brain, and the software measures the fluorescence intensity (Radiance). Under these conditions, the Luciferase (Photinus pyralis) reporter gene expressed in U118MG-luc human glioma cells can produce a fluorescent response, so the fluorescence intensity can reflect the proliferation number of glioma cells. The higher the fluorescence intensity, the greater the number of glioma cells.
  • mice in each group were administered drugs on D1, D4, D8 and D12 respectively. Weigh before administration and administer according to weight.
  • conjugate 20 was administered to each mouse respectively, and the single administration volume was 5 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 5 mg/kg, and the dose containing MMAE was equivalent to 0.3 mg/kg.
  • conjugate 21 was administered to each mouse respectively, and the single administration volume was 5 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 4 mg/kg, and the dose containing MMAE was equivalent to 0.3 mg/kg.
  • control conjugate 24 was administered to each mouse respectively, with a single administration volume of 5 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 4 mg/kg, and the dose containing MMAE was equivalent to 0.3mg/kg.
  • DMEM culture medium was given to each mouse respectively, and the single administration volume was 5 ⁇ L/g mouse body weight.
  • Figure 5 shows a line chart showing the changes in tumor fluorescence intensity over time in U118MG orthotopic tumor model mice after administration of the conjugates or control compounds provided by the present disclosure.
  • the tumor fluorescence intensity (Radiance) of the blank control group and the control group increased significantly, indicating that the number of U118MG human glioma cells increased significantly.
  • the fluorescence intensity of the test groups 5a and 5b administered with the conjugate provided by the present disclosure was significantly reduced, and the reduction was up to 1 order of magnitude, and compared with the control group, it could reach more than 2 orders of magnitude, indicating that U118MG human glue
  • the number of tumor cells is significantly reduced, and may be reduced to 1/10 of that at the beginning of the experiment, and may even be reduced to less than 1% of that of the control group. It can be seen that even if it is only administered subcutaneously, the conjugate provided by the present disclosure can effectively penetrate the blood-brain barrier and efficiently target into brain glioma, and has a good tumor growth inhibition effect, showing good treatment compliance. It has high drug potential and high efficacy in inhibiting tumors.
  • U118MG human glioma cells purchased from Guangzhou Genio Biotechnology Co., Ltd. were cultured according to the method of Experimental Example 2. Take the U118MG human glioma cells growing in the logarithmic phase, digest them with (0.25% trypsin), collect the cells, centrifuge to remove the supernatant, and resuspend the cells in serum-free DMEM medium to make a concentration of 1 ⁇ 10 8 cells/mL of cell culture medium.
  • mice 36 NOD-SCID mice (purchased from Spefford (Beijing) Biotechnology Co., Ltd.), male, 12 weeks old.
  • the above cell culture medium was inoculated subcutaneously on the right back of NOD-SCID mice, and the inoculation volume was 100 ⁇ L per mouse, that is, 1 ⁇ 10 7 cells were inoculated into each mouse. Note After injection, the mice were kept for 7 days.
  • Conjugate 20 was dissolved in serum-free DMEM medium to a conjugate solution with a concentration of 1 mg/mL (based on aptamer).
  • Conjugate 21, conjugate 22 and conjugate 23 were respectively dissolved into conjugate solutions with a concentration of 0.8 mg/mL (based on aptamer).
  • Administration began 7 days after U118MG cells were inoculated, and the day of administration was recorded as D8.
  • the experiment adopted abdominal subcutaneous administration, with administration once each on D8, D12, D16 and D20, for a total of 4 administrations.
  • DMEM was administered to each group of mice respectively, and the single administration volume was 5 ⁇ L/g mouse body weight;
  • MMAE was administered to each group of mice respectively, and the single administration volume was 5 ⁇ L/g mouse body weight. Calculation showed that the single administration dose was 0.3 mg/kg;
  • conjugate 20 was administered to each mouse respectively, and the single administration volume was 5 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 5 mg/kg, and the dose containing MMAE was equivalent to 0.3 mg/kg;
  • conjugate 21, conjugate 22 or conjugate 23 were administered to each group of mice respectively.
  • the single administration volume was 5 ⁇ L/g mouse body weight.
  • the calculation showed that the single administration dose was 4mg/kg, which contains the dose of MMAE equivalent to 0.3mg/kg; respectively recorded as test groups 6d, 6e and 6f;
  • FIG. 6 is a line graph showing the change in tumor volume over time in mice at different days after administration of different conjugates. As can be seen from Figure 6, compared with the control group 6b or the blank control group, the tumor volume and tumor weight of the mice in the test groups 6c-6f administered the conjugate of the present disclosure were significantly reduced. The above results indicate that the conjugate of the present disclosure can effectively reach tumor tissues and display good anti-tumor activity.
  • mice inoculated with U118MG subcutaneous tumors were obtained. Continue to raise the mice.
  • mice Seven days after inoculation of U118MG cells, all mice were randomly divided into 7 groups, with 6 mice in each group, and the mice in each group were administered drugs. The day of administration was recorded as D8. The experiment adopted abdominal subcutaneous administration, once each on D8, D11, D15, D29, D32 and D36, for a total of 6 administrations. Weigh before administration and calculate administration volume based on weight.
  • DMEM culture medium was given to each mouse respectively, and the single administration volume was 10 ⁇ L/g mouse body weight.
  • each mouse was given MMAE at a concentration of 0.01 mg/mL, and the single administration volume was 10 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 0.1 mg/kg.
  • each mouse was administered conjugate 21 at a concentration of 0.165 mg/mL, and the single administration volume was 10 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 1.65 mg/kg (corresponding to MMAE dose is 0.1mg/kg).
  • each mouse was administered a concentration of 0.165 mg/mL of comparative conjugate 25, and the single administration volume was 10 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 1.65 mg/kg ( The corresponding MMAE dose is 0.1 mg/kg).
  • each mouse was given MMAE at a concentration of 0.03 mg/mL.
  • the single administration volume was 10 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 0.3 mg/kg.
  • each mouse was administered conjugate 21 at a concentration of 0.5 mg/mL, and the single administration volume was 10 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 5 mg/kg (corresponding to MMAE The dose is 0.3mg/kg).
  • each mouse was given a concentration of 0.5 mg/mL of comparative conjugate 25, and the single administration volume was 10 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 5 mg/kg (corresponding to MMAE dose is 0.3 mg/kg).
  • Tumor volume was measured on D43, D47, D53, D57, D60, D64, D67, D71, D74, D78, D81, D84, D88, D92, D95 and D99.
  • the blank control group was on D53, the test groups 7a, 7b and Control group 7c was measured at D60 and the experiment was terminated.
  • the long diameter and short diameter of the tumor were determined by in vitro measurement. Tumor volume was calculated according to the formula 1/2 (long diameter ⁇ short diameter 2 ). After the experiment was terminated, tumor tissues from each group were collected, weighed, and the average value was determined. The results are shown in Figure 7.
  • Figure 7 is a line graph showing the changes in tumor volume over time in U118MG subcutaneous tumor model mice after administration of different concentrations of conjugates or control compounds provided by the present disclosure.
  • the tumor volume of test group 7b was significantly smaller than that of test group 7a and control group 7c, which were equivalent to MMAE content; during the test period, the tumor volume of test group 7e was significantly smaller than that of test group 7d and control group 7f, which were equivalent to MMAE content. , showing better anti-tumor activity than the administration of MMAE test groups 7a and 7d alone, as well as the administration of comparison conjugate 25.
  • the tumor weight in the mice administered the conjugate of the present disclosure was also significantly lower than that in the MMAE group and the control group. The above shows that the conjugate provided by the present disclosure can effectively deliver MMAE to tumor tissue. While showing tumor targeting ability, it also reduces the risk of toxicity caused by the distribution of MMAE molecules in other tissues, showing dose-related and Excellent anti-tumor effect.
  • mice inoculated with U118MG subcutaneous tumors were obtained, and the mice were continued to be raised after injection.
  • mice inoculated above Seven days after inoculation of U118MG cells, the mice inoculated above were divided into groups, with 6 mice in each group. The mice in each group were administered drugs, and the day of administration was recorded as D8. Weigh before administration, and calculate the administration volume based on the average weight of 20g per animal.
  • the experiment adopted abdominal subcutaneous administration, once each on D8, D12, D15 and D19, for a total of 4 administrations.
  • DMEM culture medium was given to each mouse respectively, with a single administration volume of 100 ⁇ L.
  • MMAE was administered to each mouse respectively, with a single administration volume of 100 ⁇ L. Calculation showed that the single administration dose was 0.1 mg/kg.
  • conjugate 20 was administered to each mouse respectively, with a single administration volume of 100 ⁇ L, and the calculation showed that the single administration dose was 1.65 mg/kg.
  • the comparison conjugate 25 was administered to each mouse respectively, with a single administration volume of 100 ⁇ L, and the calculation showed that the single administration dose was 1.65 mg/kg.
  • conjugate 21 was administered to each mouse respectively, with a single administration volume of 100 ⁇ L, and the calculation showed that the single administration dose was 1.32 mg/kg.
  • conjugate 23 was administered to each mouse respectively, with a single administration volume of 100 ⁇ L, and the calculation showed that the single administration dose was 1.32 mg/kg.
  • test group 8f conjugate 26 was administered to each mouse respectively, with a single administration volume of 100 ⁇ L, and the calculation showed that the single administration dose was 1.17 mg/kg.
  • the dosages of the above-mentioned groups 8b-8f are equivalent to containing MMAE at 0.1 mg/kg.
  • Tumor volume was measured on D1, D9, D16, D19, D22, D26, D30, D36, D40, D43, D47, D50, D54, D57, D61, D64, D68 and D71.
  • the blank control group was measured on D54, and the 8a group (only given MMAE) was measured on D64 and the experiment was terminated.
  • the long diameter and short diameter of the tumor were determined by in vitro measurement. Tumor volume was calculated according to the formula 1/2 (long diameter ⁇ short diameter 2 ). After the experiment, tumor tissues were collected from each group and weighed. The results are shown in Figure 8.
  • Figure 8 is a line graph showing changes in tumor volume over time and D72 tumor weight in each group of mice. It can be seen from the results of Figure 8 that the tumor volume of the blank control group increased rapidly, and the tumor volume increase rate of the other groups decreased; and compared with the test group 8a and the control group 8c that were only given MMAE, the conjugate of the present disclosure was given In each test group, the tumor volume increase rate was significantly further reduced when the equivalent single dose of MMAE was 0.1mg/kg.
  • Conjugate 20, Conjugate 21, Conjugate 23 and Conjugate 26 reduced the tumor weight by at least 58% compared with test group 8a at the experimental end point D72, showing more excellent anti-tumor effects.
  • the culture conditions of A549 human lung adenocarcinoma cells were DMEM complete medium (MACGENE Company, catalog number CM15019) containing 10% FBS (Gibco, catalog number 10099-141), at 37.5% Culture in a CO 2 /95% air incubator. Digest and collect the cells with 0.25wt% trypsin, aspirate the supernatant and resuspend the cells in serum-free DMEM medium to prepare a cell culture medium with a cell density of 1 ⁇ 10 8 cells/mL.
  • DMEM complete medium (MACGENE Company, catalog number CM15019) containing 10% FBS (Gibco, catalog number 10099-141)
  • FBS Gibco, catalog number 10099-141
  • mice inoculated with A549 subcutaneous tumors were obtained according to the method of Experimental Example 2, and the mice were continued to be raised after injection.
  • Conjugate 20 and conjugate 21 were dissolved in serum-free DMEM medium to a conjugate solution with a concentration of 1 mg/mL (based on aptamer).
  • Comparative conjugate 24 was dissolved into a conjugate solution with a concentration of 0.8 mg/mL (based on aptamer).
  • 10% DMSO + 90% serum-free DMEM medium (volume ratio) MMAE was dissolved into a solution with a concentration of 0.06 mg/mL.
  • mice Seven days after inoculation of A549 human lung cancer cells, all mice were divided into groups, with 6 mice in each group, and each mouse was administered a drug. The day of administration was recorded as D8. Weigh before administration, and calculate the administration volume based on mouse body weight.
  • mice in each group were administered once each on D8, D12, D15 and D19, for a total of 4 times.
  • DMEM was administered to each group of mice respectively, and the single administration volume was 5 ⁇ L/g mouse body weight;
  • MMAE was administered to each group of mice respectively, and the single administration volume was 5 ⁇ L/g mouse body weight. Calculation showed that the single administration dose was 0.3 mg/kg;
  • conjugate 20 was administered to each mouse respectively, and the single administration volume was 5 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 5 mg/kg, which contained MMAE. The dose is equivalent to 0.3mg/kg;
  • conjugate 21 was administered to each mouse respectively, and the single administration volume was 5 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 5 mg/kg, and the dose containing MMAE was equivalent to 0.3 mg/kg;
  • control conjugate 24 was administered to each mouse respectively, and the single administration volume was 5 ⁇ L/g mouse body weight. The calculation showed that the single administration dose was 5 mg/kg, and the dose containing MMAE was equivalent to 0.3mg/kg;
  • mice in each group were administered once each on D46, D50 and D54.
  • the volume of single administration was 10 ⁇ L/g mouse body weight. Calculation showed that the dose containing MMAE in a single administration was equivalent to 0.6 mg/kg.
  • Tumor volume was measured on D1, D9, D16, D19, D22, D26, D30, D36, D40, D43, D47, D50, D54 and D57.
  • the experiment was terminated after D50 measurement.
  • Figure 9 is a line graph showing the changes in tumor volume over time in A549 subcutaneous tumor model mice after administration of different concentrations of conjugates or control compounds provided by the present disclosure. It can be seen from the results in Figure 9 that the tumor volume of the mice in the blank control group increased rapidly, and the increase rate of tumor volume in the other groups decreased; at each time period, the tumor volumes of mice given conjugates 20 and 21 were all the same. smaller than controls 9b and 9e. The above results show that the conjugate of the present disclosure can effectively target and reach A549 lung cancer tumor tissue and show good anti-tumor activity.
  • any combination of various embodiments of the present disclosure can also be carried out, and as long as they do not violate the idea of the present disclosure, they should also be regarded as the contents disclosed in the present disclosure.

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Abstract

La présente divulgation concerne un conjugué. Le conjugué comprend un ou plusieurs groupes d'administration et un ou plusieurs groupes fonctionnels. Chacun des groupes d'administration est relié indépendamment au groupe fonctionnel au moyen d'une liaison covalente ou au moyen d'un groupe de liaison. Chacun des groupes fonctionnels est choisi parmi un groupe thérapeutique à petites molécules ayant un effet thérapeutique sur les tumeurs. Le conjugué fourni par la présente divulgation peut être efficacement ciblé pour une administration à des tissus tumoraux, ce qui permet de traiter efficacement des tumeurs et des maladies associées à une tumeur.
PCT/CN2023/088459 2022-04-14 2023-04-14 Conjugué et composition ainsi que leur procédé de préparation et leur utilisation WO2023198200A1 (fr)

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CN110075295A (zh) * 2013-07-23 2019-08-02 免疫医疗公司 具有cl2a接头的抗体-sn-38免疫缀合物
CN110959011A (zh) * 2017-12-29 2020-04-03 苏州瑞博生物技术有限公司 缀合物及其制备方法和用途
CN112390835A (zh) * 2019-08-14 2021-02-23 苏州瑞博生物技术股份有限公司 肝靶向化合物及缀合物

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110075295A (zh) * 2013-07-23 2019-08-02 免疫医疗公司 具有cl2a接头的抗体-sn-38免疫缀合物
CN110959011A (zh) * 2017-12-29 2020-04-03 苏州瑞博生物技术有限公司 缀合物及其制备方法和用途
CN112390835A (zh) * 2019-08-14 2021-02-23 苏州瑞博生物技术股份有限公司 肝靶向化合物及缀合物

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